CDC7 kinase inhibitors and uses thereof

ABSTRACT

The invention provides compounds, methods, pharmaceutical compositions, and kits for the treatment of proliferative disorders such as cancer. In one aspect, the methods comprise compounds that inhibit the activity of protein kinases, such as cell division cycle (Cdc) kinase. In another aspect, the methods comprise compounds that inhibit Cdc7 and/or Dbf4 activity. In another aspect, the methods comprise compounds that exhibit anti-proliferative properties useful in treating diseases such as cancer. Compounds useful for any of the methods include compounds of the Formula (A) or (B): 
                         
or pharmaceutically acceptable salts thereof. Exemplary compounds of Formula (A) or (B) include granaticin A, granaticin B, dihydrogranaticin A, dihydrogranaticin B, medermycin, and actinorhodin.

RELATED APPLICATIONS

The present application is a continuation of and claims priority under35 U.S.C. §120 to U.S. application Ser. No. 14/936,472, filed Nov. 9,2015, now U.S. Pat. No. 9,492,427, which is a continuation of and claimspriority under 35 U.S.C. §120 to U.S. application Ser. No. 13/583,170,filed Oct. 18, 2012, now U.S. Pat. No. 9,180,105, which is a nationalstage filing under 35 U.S.C. §371 of international PCT application,PCT/US2011/027619, filed Mar. 8, 2011, which claims priority under 35U.S.C. §119(e) to U.S. provisional patent application, U.S. Ser. No.61/311,741, filed Mar. 8, 2010, each of which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

Understanding how the genomes of eukaryotes are duplicated during eachcell cycle is a fundamental problem of modern biology and is a criticalaspect of the more general problem of understanding the mechanisms thatcontrol cellular proliferation. The transition from G1 into S phase is amajor decision point for the cell and is subject to elaborate controlswhose mechanisms are not yet understood at the molecular level. (Bell,S. P. and A. Dutta (2002) Annu Rev Biochem 71: 333-74; Dutta, A. and S.P. Bell (1997) Annu Rev Cell Dev Biol 13: 293-332; Jallepalli, P. V. andT. J. Kelly (1997) Curr Opin Cell Biol 9(3): 358-63; Kelly, T. J. and G.W. Brown (2000) AnnuRev Biochem 69: 829-80; Stillman, B. (1996) Science274(5293):1659-64) The stability of the genome depends upon the preciseoperation of the DNA “replication switch,” as well as upon the propercoupling of DNA replication to other events in the cell. It has becomequite clear that perturbation of any of these mechanisms can contributeto cancer. (Sherr, C. J. (1996). Science 274(5293): 1672-7)

During the last decade, work in a number of laboratories has led to adramatic advance in our understanding of cellular DNA replication (Bell,S. P. and A. Dutta, et al.; Kelly, T. J. and G. W. Brown, et al.). Theanalysis of simple model systems, particularly Saccharomyces cerevisiae,Schizosaccharomyces pombe, and Xenopus laevis, has resulted in theidentification of proteins that act at origins of DNA replication toinitiate DNA synthesis. A significant breakthrough was the discovery byStillman and Bell of the six-subunit origin recognition complex (ORC),which binds to specific origins of DNA replication in S. cerevisiae andrecruits additional initiation factors to form the pre-replicationcomplex (pre-RC). The ORC has been conserved throughout eukaryoticevolution. (Chuang, R. Y., L. Chretien, et al. (2002) J Biol Chem277(19): 16920-7; Gossen, M., D. T. Pak, et al. (1995) Science270(5242): 1674-7; Moon, K. Y., D. Kong, et al. (1999) Proc Natl AcadSci USA 96(22): 12367-12372; Rowles, A., J. P. Chong, et al. (1996) Cell87(2): 287-96; Vashee, S., P. Simancek, et al. (2001) J Biol Chem276(28): 26666-73) We now know that a common set of initiation proteinsassemble at replication origins in all eukaryotes and that theactivities of these proteins are regulated by specific protein kinases.However, despite this progress, our understanding of the biochemicalmechanisms of initiation of eukaryotic DNA replication remains quitesuperficial.

Genetic studies in yeasts and biochemical studies in Xenopus havedemonstrated that the initiation of eukaryotic DNA replication takesplace in two stages. (Bell, S. P. and A. Dutta, et al.; Kelly, T. J. andG. W. Brown, et al.) In the first stage, which lasts from late M throughthe G1 phase of the cell cycle, pre-RCs are assembled at origins of DNAreplication. At the beginning of S phase, pre-RCs are activated by theaction of two heterodimeric protein kinases, Cdc7-Dbf4 and S phasecyclin-dependent kinase (S-CDK). This event marks the transition to thesecond stage of initiation, during which the origin is unwound andadditional proteins are recruited to form active replication forks. Thepresence of cyclin dependent kinase activity (and perhaps otherinhibitory factors) prevents further assembly of pre-RCs during thesecond stage of the initiation reaction. This mechanism constitutes a“replication switch” that ensures that origins of DNA replication fireonly once each cell cycle, thus preserving genomic integrity.

As noted above, the activation of the pre-RC requires the activities ofCdc7-Dbf4 and S-CDK. Both kinases are activated at the G1/S boundarywhen their respective regulatory subunits accumulate to sufficientlevels, and both appear to associate with the pre-RC. (Brown, G. W., P.V. Jallepalli, et al. (1997) Proc. Natl. Acad. Sci., USA 94: 6142-6147;Dowell, S. J., P. Romanowski, et al. (1994) Science 265(5176): 1243-6;Jallepalli, P. V. and T. J. Kelly; Jares, P. and J. J. Blow (2000) GenesDev 14(12): 528-40; Johnston, L. H., H. Masai, et al. (1999) Trends CellBiol 9(7): 249-52; Leatherwood, J., A. Lopez-Girona, et al. (1996)Nature 379(6563): 360-3; Walter, J. C. (2000) J. Biol. Chem. 275(50):39773-8) Although the regulation of S-CDK activity has been shown to bequite complex with multiple cyclin subunits pairing with multiple Cdksubunits, Cdc7 activity is strictly regulated by the expression of theDbf4 subunit, which is very tightly cell cycle regulated with peakexpression occurring at the G1/S boundary. (Bell, S. P. and A. Dutta, etal.) The activity of Cdc7 has been shown to be required for entry into Sphase of the cell cycle. Studies in yeast have shown that cells depletedof this kinase activity progress from G1 to M phase without anintervening S phase, resulting in cell death (Bell, S. P. and A. Dutta,et al.; Kelly, T. J. and G. W. Brown, et al.), and conditional knockoutmouse Embryonic stem (ES) cells for Dbf4 have recently been shown toundergo S phase arrest with resultant apoptosis when gene expression issilenced. (Yamashita, N., Kim, J-M, et al. (2005) Genes to Cells 10:551-563) Genetic evidence has shown that the six subunit MinichromosomeMaintenance complex (MCM2-7), the presumed helicase activity requiredfor origin unwinding and the initiation of DNA replication (Bell, S. P.and A. Dutta, et al.; Kelly, T. J. and G. W. Brown, et al.), is a targetof regulation by the Cdc7-Dbf4 kinase, and the Mcm2 protein is anexcellent substrate for the Cdc7:Dbf4 kinase in vitro. (Sclafani, R. A.(2000) J Cell Sci 113 (Pt 12): 2111-7) The MCM proteins and Cdc7 havebeen shown to be overexpressed in the majority of cancers including bothsolid tumors and hematologic malignancies. (Hess, G. F., Drong, R. F.,et al. (1998) Gene 211 (1):133-40; Velculescu, V. E., Madden, S. L., etal. (1999) Nature Genetics 23: 387-88) Importantly, it has recently beenshown that overexpression of Cdc7 in cutaneous melanoma samples wasassociated with poor risk disease and chemotherapy resistance. (Nambiar,S., Mirmohammadsadegh, A., et al. (2007) Carcinogenesis 12: 2501-2510)In addition, Cdc7 overexpression has also been shown in aggressiveundifferentiated papillary thyroid carcinoma and in aggressive head andneck cancers that are positive for human papillomavirus (Fluge, O.,Bruland, O., Akslen, L. A., et al. (2006) Thyroid 16 (2): 161-175;Slebos, R. J. C, Yi, Y., Ely, K., et al. (2006) Clin Cancer Res 12(3):701-709). In fact, sensitive assay systems are being developed in Europeand the United States to detect the presence of MCM proteins in theurine of patients with genitourinary malignancies as well as breastcancer patients, and this seems to correlate with a more aggressivemalignancy. Cdc7 activity is also conserved from yeast to man making itan attractive candidate for a therapeutic target. The logicalinterpretation of this data is that Cdc7:Dbf4 is a bona fide therapeutictarget.

SUMMARY OF THE INVENTION

The present invention originates from an appreciation for the criticalroles of protein kinases, such as Cdc7, and the discovery that a knownclass of compounds, the benzoisochromanequinones (BIQs), such as thegranaticins, inhibit protein kinase activity. The granaticins, inparticular, were discovered to inhibit Cdc kinase activity based uponhits identified from high-thoroughput screening (HTS) of over 300,000compounds for their ability to inhibit a heterodimer of a kinase (Cdc7)and an activator (Dbf4) that phosphorylates serine and threonineresidues. It was further discovered that the granaticins may exhibitproperties useful in treating proliferative disorders, such as cancer.

The granaticins are members of a class of Streptomyces aromaticantibiotics known as benzoisochromanequinones (BIQs; Ichinose et al.,Actinomycetologica (1998) 12: 99-109), which also includes medermycin(also known as lactoquinomycin A) and actinorhodin. Of particularinterest are chemical modifications at C-10 via a C—C bond either byglycosylation (medermcyin and granaticin) or by dimerization (inactinorhodin) (see, e.g., Hopwood, Chem Rev (1997) 97: 2465-2497; Flosset al., J Nat Prod. (1986) 49:957-70, Toral-Barza et al., Mol. CancerTher. (2007) 6:3028-3038; and Salaski et al., J. Med. Chem. (2009)23:2181-2184), or by chemical or enzymatic modification of the 8-diol ofthe 3-methyl-2-oxabicyclo[2.2.2]oct-5-ene-4,8-diol group of granaticin A(e.g., coupling a carbohydrate or sugar moiety), e.g., to providecompounds such as granaticin B. The benzoisochromanequinones,granaticins, and related natural products are incorporated into themethods of use, pharmaceutical compositions, and kits described herein.

In one aspect, the present invention is directed to compounds of theFormula (A) or (B):

or a pharmaceutically acceptable salt thereof,wherein:

each instance of R¹ and R⁴ is independently selected from the groupconsisting of hydrogen, carbonyl, silyl, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl;

each instance of R² and R³ is independently selected from the groupconsisting of hydrogen, halogen, —OH, substituted hydroxyl, —SH,substituted thiol, —NH₂, substituted amino, —CN, —NO₂, carbonyl, silyl,sulfinyl, sulfonyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, and optionally substituted heteroaryl; or R² and R³ are joined toform an optionally substituted carbocyclyl, optionally substitutedheterocyclyl, optionally substituted aryl, or optionally substitutedheteroaryl group;

R⁵ is hydrogen and R⁶ is selected from the group consisting of hydrogen,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl; or R⁵ and R⁶ are joined to form a direct bond;

R⁷ is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, and optionally substituted heteroaryl;

R¹² is hydrogen, carbonyl, silyl, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl; and

each instance of R¹³ and R¹⁴ is independently selected from the groupconsisting of hydrogen, halogen, —OH, substituted hydroxyl, —SH,substituted thiol, —NH₂, substituted amino, —CN, —NO₂, carbonyl, silyl,sulfinyl, sulfonyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, and optionally substituted heteroaryl; or R¹³ and R¹⁴ are joinedto form an optionally substituted carbocyclyl, optionally substitutedheterocyclyl, optionally substituted aryl, or optionally substitutedheteroaryl group;

for use in treating or preventing a proliferative disorder.

In another aspect, the present invention provides use of a compound ofFormula (A) or (B), or a pharmaceutically acceptable salt thereof, forthe manufacture of a medicament for treating or preventing aproliferative disorder.

In yet another aspect, the present invention provides a method fortreating or preventing a proliferative disorder in a subject in needthereof, wherein the subject is administered a therapeutically effectiveamount of a compound of the Formula (A) or (B), or a pharmaceuticallyacceptable salt thereof.

In certain embodiments, the compound is of the Formula (A-3):

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the compound is of the Formula (A-7):

or a pharmaceutically acceptable salt thereof,

wherein each instance of R⁹ and R¹⁰ is independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, carbonyl, silyl, sulfonyl, andsulfinyl.

In certain embodiments, the compound is of the Formula (B-1):

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the compound is selected from the groupconsisting of:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the compound is an inhibitor of a proteinkinase; and wherein the inhibition of a protein kinase is useful for thetreatment or prevention of the proliferative disorder. In certainembodiments, the protein kinase is Cdc7 kinase or the Dbf4 regulatorysubunit of Cdc7 kinase, and wherein the inhibition of Cdc7 kinase or theDbf4 regulatory subunit of Cdc7 kinase is useful for the treatment orprevention of the proliferative disorder.

In certain embodiments, the proliferative disorder is selected from thegroup consisting of cancer, myeloproliferative disorders, benignprostate hyperplasia, familial adenomatosis, polyposis,neuro-fibromatosis, psoriasis, vascular smooth cell proliferationassociated with atherosclerosis, fibrotic disorders, pulmonary fibrosis,arthritis, rheumatoid arthritis, glomerulonephritis, and post-surgicalstenosis, restenosis, disorders of proliferation of blood vessels,disorders of proliferation of mesangial cells, metabolic disorders,allergies, asthmas, thromboses, diseases of the nervous system,retinopathy, diabetes, and muscular degeneration. In certainembodiments, proliferative disorder is cancer.

In certain embodiments, the cancer is selected from the group consistingof bone, brain, connective tissue, endocrine glands, adrenal cortex,endometrium, germ cells, head and neck, larynx and hypopharynx,mesothelioma, muscle, rectum, renal, small intestine, soft tissue,testis, ureter, vagina, and vulva; bladder cancer; breast cancer; coloncancer; kidney cancer; liver cancer; lung cancer; esophagus cancer;gallbladder cancer; ovarian cancer; pancreatic cancer; stomach cancer;cervical cancer; thyroid cancer; prostate cancer; papillary thyroidcarcinoma; genitourinary malignancies; retinoblastoma; Wilms tumor;myelodysplastic syndrome; plasma cell neoplasia; paraneoplasticsyndromes; renal cell carcinoma; Ewing's sarcoma; desmoplastic smallround cell tumors; mesothelioma; skin cancer, wherein said skin canceris squamous cell carcinoma; hematologic cancers [e.g., hematopoieticcancers of lymphoid lineage, wherein said cancers are leukemia, acutelymphocytic leukemia (ALL), acute lymphoblastic leukemia, lymphoma,B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin'slymphoma (e.g., mantle cell lymphoma (MCL), hairy cell lymphoma, andBurkitt's lymphoma; chronic lymphocytic leukemia (CLL); hematopoieticcancers of myeloid lineage, wherein said cancers are multiple myeloma,chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) (e.g.,acute megakaryoblastic leukemia (AMKL); myelodysplastic syndrome andpromyelocytic leukemia]; tumors of mesenchymal origin, wherein thetumors are fibr osarcoma and rhabdomyosarcoma; tumors of the central andperipheral nervous system, wherein said tumors are astrocytoma,neuroblastoma, glioma (e.g., glioblastoma) and schwannomas; and othertumors, wherein said tumors are melanoma, cutaneous melanoma, seminoma,teratocarcinoma, osteosarcoma, xeroderma pegmentosum, keratoxanthoma,thyroid follicular cancer, Kaposi's sarcoma, cancers of unknown primarysite; solid tumors, hematologic cancers, and AIDS-related malignancies.

In certain embodiments, the cancer is a hematologic cancer, ovariancancer, lung cancer, prostate cancer, renal cell carcinoma, cervicalcancer, glioblastoma, retinoblastoma, rhabdomyosarcoma, desmoplasticsmall round cell tumor, breast cancer, mesothelioma, melanoma, thyroidcarcinoma, Ewing's sarcoma, or a solid tumor.

In certain embodiments, the cancer comprises a genetic mutation. Incertain embodiments, the genetic mutation comprises a RAS mutation, anEGFR mutation, a KRAS mutation, a p53 mutation, a BRAF mutation, a EVI1mutation, a Flt-3 mutation, WT-1 mutation, cyclin D mutation, PTENmutation, ABL kinase mutation, or a chromosomal abnormality.

In certain embodiments, the cancer is a multi-drug resistant (MDR)cancer.

In certain embodiments, the cancer is relapsed and/or refractory cancer.

In certain embodiments, the method further comprises administering atleast one other therapy or therapeutic agent. In certain embodiments,the method further comprises administering radiation.

Also provided are pharmaceutical composition and kits useful in any ofthe methods.

The details of one or more embodiments of the invention are set forth inthe accompanying Figures and the Detailed Description. Other features,objects, and advantages of the invention will be apparent from thedescription and from the claims.

Definitions

Compounds useful in the present invention include inhibitors of Cdc7kinase and/or Dbf4. Definitions of specific functional groups andchemical terms are described in more detail below. The chemical elementsare identified in accordance with the Periodic Table of the Elements,CAS version, Handbook of Chemistry and Physics, 75^(th) Ed., insidecover, and specific functional groups are generally defined as describedtherein. Additionally, general principles of organic chemistry, as wellas specific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March March's Advanced Organic Chemistry, 5^(th) Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various isomeric forms, e.g., enantiomers and/ordiastereomers. For example, the compounds described herein can be in theform of an individual enantiomer, diastereomer or geometric isomer, orcan be in the form of a mixture of stereoisomers, including racemicmixtures and mixtures enriched in one or more stereoisomer. Isomers canbe isolated from mixtures by methods known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or preferred isomers canbe prepared by asymmetric syntheses. See, for example, Jacques et al.,Enantiomers, Racemates and Resolutions (Wiley Interscience, New York,1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E. L.Stereochemistry of Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen,S. H. Tables of Resolving Agents and Optical Resolutions p. 268 (E. L.Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972). Theinvention additionally encompasses compounds as individual isomerssubstantially free of other isomers, and alternatively, as mixtures ofvarious isomers.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

As used herein, “alkyl” refers to a radical of a straight-chain orbranched saturated hydrocarbon group having from 1 to 10 carbon atoms(“C₁₋₁₀ alkyl”). In some embodiments, an alkyl group has 1 to 9 carbonatoms (“C₁₋₉ alkyl”). In some embodiments, an alkyl group has 1 to 8carbon atoms (“C₁₋₈ alkyl”). In some embodiments, an alkyl group has 1to 7 carbon atoms (“C₁₋₇ alkyl”). In some embodiments, an alkyl grouphas 1 to 6 carbon atoms (“C₁₋₆ alkyl”). In some embodiments, an alkylgroup has 1 to 5 carbon atoms (“C₁₋₅ alkyl”). In some embodiments, analkyl group has 1 to 4 carbon atoms (“C₁₋₄ alkyl”). In some embodiments,an alkyl group has 1 to 3 carbon atoms (“C₁₋₃ alkyl”). In someembodiments, an alkyl group has 1 to 2 carbon atoms (“C₁₋₂ alkyl”). Insome embodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). In someembodiments, an alkyl group has 2 to 6 carbon atoms (“C₂₋₆ alkyl”).Examples of C₁₋₆ alkyl groups include methyl (C₁), ethyl (C₂), n-propyl(C₃), isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄),iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl(C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅), and n-hexyl (C₆).Additional examples of alkyl groups include n-heptyl (C₇), n-octyl (C₈)and the like. Unless otherwise specified, each instance of an alkylgroup is independently unsubstituted (an “unsubstituted alkyl”) orsubstituted (a “substituted alkyl”) with one or more substituents. Incertain embodiments, the alkyl group is an unsubstituted C₁₋₁₀ alkyl(e.g., —CH₃). In certain embodiments, the alkyl group is a substitutedC₁₋₁₀ alkyl.

“Perhaloalkyl” is a substituted alkyl group as defined herein whereinall of the hydrogen atoms are independently replaced by a halogen, e.g.,fluoro, bromo, chloro, or iodo. In some embodiments, the alkyl moietyhas 1 to 8 carbon atoms (“C₁₋₈ perhaloalkyl”). In some embodiments, thealkyl moiety has 1 to 6 carbon atoms (“C₁₋₆ perhaloalkyl”). In someembodiments, the alkyl moiety has 1 to 4 carbon atoms (“C₁₋₄perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 3 carbonatoms (“C₁₋₃ perhaloalkyl”). In some embodiments, the alkyl moiety has 1to 2 carbon atoms (“C₁₋₂ perhaloalkyl”). In some embodiments, all of thehydrogen atoms are replaced with fluoro. In some embodiments, all of thehydrogen atoms are replaced with chloro. Examples of perhaloalkyl groupsinclude —CF₃, —CF₂CF₃, —CF₂CF₂CF₃, —CCl₃, —CFCl₂, —CF₂Cl, and the like.

As used herein, “alkenyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 10 carbon atoms and one ormore carbon-carbon double bonds (“C₂₋₁₀ alkenyl”). In some embodiments,an alkenyl group has 2 to 9 carbon atoms (“C₂₋₉ alkenyl”). In someembodiments, an alkenyl group has 2 to 8 carbon atoms (“C₂₋₈ alkenyl”).In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C₂₋₇alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms(“C₂₋₆ alkenyl”). In some embodiments, an alkenyl group has 2 to 5carbon atoms (“C₂₋₅ alkenyl”). In some embodiments, an alkenyl group has2 to 4 carbon atoms (“C₂₋₄ alkenyl”). In some embodiments, an alkenylgroup has 2 to 3 carbon atoms (“C₂₋₃ alkenyl”). In some embodiments, analkenyl group has 2 carbon atoms (“C₂ alkenyl”). The one or morecarbon-carbon double bonds can be internal (such as in 2-butenyl) orterminal (such as in 1-butenyl). Examples of C₂₋₄ alkenyl groups includeethenyl (C₂), 1-propenyl (C₃), 2-propenyl (C₃), 1-butenyl (C₄),2-butenyl (C₄), butadienyl (C₄), and the like. Examples of C₂₋₆ alkenylgroups include the aforementioned C₂₋₄ alkenyl groups as well aspentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and the like. Additionalexamples of alkenyl include heptenyl (C₇), octenyl (C₈), octatrienyl(C₈), and the like. Unless otherwise specified, each instance of analkenyl group is independently unsubstituted (an “unsubstitutedalkenyl”) or substituted (a “substituted alkenyl”) with one or moresubstituents. In certain embodiments, the alkenyl group is anunsubstituted C₂₋₁₀ alkenyl. In certain embodiments, the alkenyl groupis a substituted C₂₋₁₀ alkenyl.

As used herein, “alkynyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 10 carbon atoms and one ormore carbon-carbon triple bonds (“C₂₋₁₀ alkynyl”). In some embodiments,an alkynyl group has 2 to 9 carbon atoms (“C₂₋₉ alkynyl”). In someembodiments, an alkynyl group has 2 to 8 carbon atoms (“C₂₋₈ alkynyl”).In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C₂₋₇alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms(“C₂₋₆ alkynyl”). In some embodiments, an alkynyl group has 2 to 5carbon atoms (“C₂₋₅ alkynyl”). In some embodiments, an alkynyl group has2 to 4 carbon atoms (“C₂₋₄ alkynyl”). In some embodiments, an alkynylgroup has 2 to 3 carbon atoms (“C₂₋₃ alkynyl”). In some embodiments, analkynyl group has 2 carbon atoms (“C₂ alkynyl”). The one or morecarbon-carbon triple bonds can be internal (such as in 2-butynyl) orterminal (such as in 1-butynyl). Examples of C₂₋₄ alkynyl groupsinclude, without limitation, ethynyl (C₂), 1-propynyl (C₃), 2-propynyl(C₃), 1-butynyl (C₄), 2-butynyl (C₄), and the like. Examples of C₂₋₆alkenyl groups include the aforementioned C₂₋₄ alkynyl groups as well aspentynyl (C₅), hexynyl (C₆), and the like. Additional examples ofalkynyl include heptynyl (C₇), octynyl (C₈), and the like. Unlessotherwise specified, each instance of an alkynyl group is independentlyunsubstituted (an “unsubstituted alkynyl”) or substituted (a“substituted alkynyl”) with one or more substituents. In certainembodiments, the alkynyl group is an unsubstituted C₂₋₁₀ alkynyl. Incertain embodiments, the alkynyl group is a substituted C₂₋₁₀ alkynyl.

As used herein, “carbocyclyl” refers to a radical of a non-aromaticcyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C₃₋₁₀carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. Insome embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms(“C₃₋₈ carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to6 ring carbon atoms (“C₃₋₆ carbocyclyl”). In some embodiments, acarbocyclyl group has 3 to 6 ring carbon atoms (“C₃₋₆ carbocyclyl”). Insome embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms(“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groups include,without limitation, cyclopropyl (C₃), cyclopropenyl (C₃), cyclobutyl(C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅),cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and the like.Exemplary C₃₋₈ carbocyclyl groups include, without limitation, theaforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇),cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇),bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclylgroups include, without limitation, the aforementioned C₃₋₈ carbocyclylgroups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀),cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl(C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing afused, bridged or spiro ring system such as a bicyclic system (“bicycliccarbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can besaturated or can contain one or more carbon-carbon double or triplebonds. “Carbocyclyl” also includes ring systems wherein the carbocyclylring, as defined above, is fused with one or more aryl or heteroarylgroups wherein the point of attachment is on the carbocyclyl ring, andin such instances, the number of carbons continue to designate thenumber of carbons in the carbocyclic ring system. Unless otherwisespecified, each instance of a carbocyclyl group is independentlyunsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is an unsubstituted C₃₋₁₀carbocyclyl. In certain embodiments, the carbocyclyl group is asubstituted C₃₋₁₀ carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 10 ring carbon atoms (“C₃₋₁₀cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ringcarbon atoms (“C₃₋₈ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 6 ring carbon atoms (“C₃₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C₅₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ cycloalkyl”). Examples of C₅₋₆ cycloalkyl groupsinclude cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C₃₋₆cycloalkyl groups include the aforementioned C₅₋₆ cycloalkyl groups aswell as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C₃₋₈cycloalkyl groups include the aforementioned C₃₋₆ cycloalkyl groups aswell as cycloheptyl (C₇) and cyclooctyl (C₈). Unless otherwisespecified, each instance of a cycloalkyl group is independentlyunsubstituted (an “unsubstituted cycloalkyl”) or substituted (a“substituted cycloalkyl”) with one or more substituents. In certainembodiments, the cycloalkyl group is an unsubstituted C₃₋₁₀ cycloalkyl.In certain embodiments, the cycloalkyl group is a substituted C₃₋₁₀cycloalkyl.

As used herein, “heterocyclyl” refers to a radical of a 3- to14-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). Inheterocyclyl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or polycyclic (e.g., a fused, bridged or spiro ring system such as abicyclic system (“bicyclic heterocyclyl”) or tricyclic system(“tricyclic heterocyclyl”)), and can be saturated or can contain one ormore carbon-carbon double or triple bonds. Heterocyclyl polycyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclyl ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclylring, or ring systems wherein the heterocyclyl ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclyl ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclyl ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with one or more substituents. In certain embodiments,the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl.In certain embodiments, the heterocyclyl group is a substituted 3-14membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-8 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl groupis a 5-6 membered non-aromatic ring system having ring carbon atoms and1-4 ring heteroatoms, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In someembodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclylhas 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary4-membered heterocyclyl groups containing 1 heteroatom include, withoutlimitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5-memberedheterocyclyl groups containing 1 heteroatom include, without limitation,tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl,dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2,5-dione.Exemplary 5-membered heterocyclyl groups containing 2 heteroatomsinclude, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl.Exemplary 5-membered heterocyclyl groups containing 3 heteroatomsinclude, without limitation, triazolinyl, oxadiazolinyl, andthiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1heteroatom include, without limitation, piperidinyl, tetrahydropyranyl,dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groupscontaining 2 heteroatoms include, without limitation, piperazinyl,morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclylgroups containing 2 heteroatoms include, without limitation,triazinanyl. Exemplary 7-membered heterocyclyl groups containing 1heteroatom include, without limitation, azepanyl, oxepanyl andthiepanyl. Exemplary 8-membered heterocyclyl groups containing 1heteroatom include, without limitation, azocanyl, oxecanyl andthiocanyl. Exemplary bicyclic heterocyclyl groups include, withoutlimitation, indolinyl, isoindolinyl, dihydrobenzofuranyl,dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl,tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl,octahydroisochromenyl, decahydronaphthyridinyl,decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl,phthalimidyl, naphthalimidyl, chromanyl, chromenyl,1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl,5,7-dihydro-4H-thieno[2,3-c]pyranyl,2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl,4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl,4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

As used herein, “aryl” refers to a radical of a monocyclic or polycyclic(e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6,10, or 14 r electrons shared in a cyclic array) having 6-14 ring carbonatoms and zero heteroatoms provided in the aromatic ring system (“C₆₋₁₄aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C₆aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ringcarbon atoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms(“C₁₋₄ aryl”; e.g., anthracyl). “Aryl” also includes ring systemswherein the aryl ring, as defined above, is fused with one or morecarbocyclyl or heterocyclyl groups wherein the radical or point ofattachment is on the aryl ring, and in such instances, the number ofcarbon atoms continue to designate the number of carbon atoms in thearyl ring system. Unless otherwise specified, each instance of an arylgroup is independently unsubstituted (an “unsubstituted aryl”) orsubstituted (a “substituted aryl”) with one or more substituents. Incertain embodiments, the aryl group is an unsubstituted C₆₋₁₄ aryl. Incertain embodiments, the aryl group is a substituted C₆₋₁₄ aryl.

“Aralkyl” is a subset of “alkyl” and refers to an alkyl group, asdefined herein, substituted by an aryl group, as defined herein, whereinthe point of attachment is on the alkyl moiety.

As used herein, “heteroaryl” refers to a radical of a 5-14 memberedmonocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromaticring system (e.g., having 6, 10, or 14π electrons shared in a cyclicarray) having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen and sulfur (“5-14 membered heteroaryl”). Inheteroaryl groups that contain one or more nitrogen atoms, the point ofattachment can be a carbon or nitrogen atom, as valency permits.Heteroaryl polycyclic ring systems can include one or more heteroatomsin one or both rings. “Heteroaryl” includes ring systems wherein theheteroaryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the point of attachment is on theheteroaryl ring, and in such instances, the number of ring memberscontinue to designate the number of ring members in the heteroaryl ringsystem. “Heteroaryl” also includes ring systems wherein the heteroarylring, as defined above, is fused with one or more aryl groups whereinthe point of attachment is either on the aryl or heteroaryl ring, and insuch instances, the number of ring members designates the number of ringmembers in the fused polycyclic (aryl/heteroaryl) ring system.Polycyclic heteroaryl groups wherein one ring does not contain aheteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) thepoint of attachment can be on either ring, i.e., either the ring bearinga heteroatom (e.g., 2-indolyl) or the ring that does not contain aheteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unlessotherwise specified, each instance of a heteroaryl group isindependently unsubstituted (an “unsubstituted heteroaryl”) orsubstituted (a “substituted heteroaryl”) with one or more substituents.In certain embodiments, the heteroaryl group is an unsubstituted 5-14membered heteroaryl. In certain embodiments, the heteroaryl group is asubstituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include,without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary5-membered heteroaryl groups containing 2 heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing 3heteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4heteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing 1 heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, andpyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4heteroatoms include, without limitation, triazinyl and tetrazinyl,respectively. Exemplary 7-membered heteroaryl groups containing 1heteroatom include, without limitation, azepinyl, oxepinyl, andthiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, withoutlimitation, indolyl, isoindolyl, indazolyl, benzotriazolyl,benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl,benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl,benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, andpurinyl. Exemplary 6,6-bicyclic heteroaryl groups include, withoutlimitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplarytricyclic heteroaryl groups include, without limitation,phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl,phenoxazinyl and phenazinyl.

“Heteroaralkyl” is a subset of “alkyl” and refers to an alkyl group, asdefined herein, substituted by a heteroaryl group, as defined herein,wherein the point of attachment is on the alkyl moiety.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aromatic groups (e.g., arylor heteroaryl moieties) as herein defined.

As used herein, a “direct bond” refers to the direct attachment of agroup via a single bond.

Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylgroups, as defined herein, are optionally substituted (e.g.,“substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted”alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or“unsubstituted” carbocyclyl, “substituted” or “unsubstituted”heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or“unsubstituted” heteroaryl group). In general, the term “substituted”,whether preceded by the term “optionally” or not, means that at leastone hydrogen present on a group (e.g., a carbon or nitrogen atom) isreplaced with a permissible substituent, e.g., a substituent which uponsubstitution results in a stable compound, e.g., a compound which doesnot spontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction. Unless otherwise indicated,a “substituted” group has a substituent at one or more substitutablepositions of the group, and when more than one position in any givenstructure is substituted, the substituent is either the same ordifferent at each position. The term “substituted” is contemplated toinclude substitution with all permissible substituents of organiccompounds, any of the substituents described herein that results in theformation of a stable compound. The present invention contemplates anyand all such combinations in order to arrive at a stable compound. Forpurposes of this invention, heteroatoms such as nitrogen may havehydrogen substituents and/or any suitable substituent as describedherein which satisfy the valencies of the heteroatoms and results in theformation of a stable moiety.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂,—N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa),—SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂, —CO₂R^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa),—NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR^(aa), —OSO₂R^(aa),—S(═O)R^(aa), —OS(═O)R^(aa), —Si(R^(aa))₃, —OSi(R^(aa))₃,—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa), —SC(═S)SR^(a),—SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa), —SC(═O)R^(aa),—P(═O)₂R^(aa), —OP(═O)₂R^(aa), —P(═O)(R^(aa))₂, —OP(═O)(R^(aa))₂,—OP(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, —OP(═O)₂N(R^(bb))₂,—P(═O)(NR^(bb))₂, —OP(═O)(NR^(bb))₂, —NR^(bb)P(═O)(OR^(cc))₂,—NR^(bb)P(═O)(NR^(bb))₂, —P(R^(cc))₂, —P(R^(cc))₃, —OP(R^(cc))₂,—OP(R^(cc))₃, —B(R^(aa))₂, —B(OR^(cc))₂, —BR^(aa)(OR^(cc)), C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₄ carbocyclyl,3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl,wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R^(dd) groups;

or two geminal hydrogens on a carbon atom are replaced with the group═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa), ═NNR^(bb)C(═O)OR^(aa),═NNR^(bb)S(═O)₂R^(aa), ═NR^(bb), or ═NOR^(cc);

each instance of R^(aa) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl,3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, ortwo R^(aa) groups are joined to form a 3-14 membered heterocyclyl or5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(bb) is, independently, selected from hydrogen, —OH,—OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(a), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂,—SO₂R^(cc), —SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc),—C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂,—P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, or two R^(bb) groups are joined to form a 3-14membered heterocyclyl or 5-14 membered heteroaryl ring, wherein eachalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylis independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(cc) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(dd) is, independently, selected from halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R^(ff))₂, —N(R^(ff))₂,—N(R^(ff))₃ ⁺X⁻, —N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee),—C(═O)R^(ee), —CO₂H, —CO₂R^(ee), —OC(═O)R^(ee), —OCO₂R^(ee),—C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂, —NR^(ff)C(═O)R^(ee),—NR^(ff)CO₂R^(ee), —NR^(ff)C(═O)N(R^(ff))₂, —C(═NR^(ff))OR^(ee),—OC(═NR^(ff))R^(ee), OC(═NR^(ff))OR^(ee), —C(═NR^(ff))N(R^(ff))₂,—OC(═NR^(ff))N(R^(ff))₂, —NR^(ff)C(═NR^(ff))N(R^(ff))₂,—NR^(ff)SO₂R^(ee), —SO₂N(R^(ff))₂, —SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee),—S(═O)R^(ee), —Si(R^(ee))₃, —OSi(R^(ee))₃, —C(═S)N(R^(ff))₂,—C(═O)SR^(ee), —C(═S)SR^(ee), —SC(═S)SR^(ee), —P(═O)₂R^(ee),—P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂, —OP(═O)(OR^(ee))₂, C₁₋₆ alkyl, C₁₋₆perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, 3-10membered heterocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, whereineach alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg)groups, or two geminal R^(dd) substituents can be joined to form ═O or═S;

each instance of R^(ee) is, independently, selected from C₁₋₆ alkyl,C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, whereineach alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg)groups;

each instance of R^(ff) is, independently, selected from hydrogen, C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl,3-10 membered heterocyclyl, C₆₋₁₀ aryl and 5-10 membered heteroaryl, ortwo R^(ff) groups are joined to form a 3-14 membered heterocyclyl or5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups; and

each instance of R^(gg) is, independently, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₂,—N(C₁₋₆ alkyl)₃ ⁺X⁻, —NH(C₁₋₆ alkyl)₂ ⁺X⁻, —NH₂(C₁₋₆ alkyl)⁺X⁻, —NH₃⁺X⁻, —N(OC₁₋₆ alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆ alkyl), —NH(OH), —SH,—SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl), —C(═O)(C₁₋₆ alkyl), —CO₂H, —CO₂(C₁₋₄alkyl), —OC(═O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆ alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆alkyl)₂, —OC(═O)NH(C₁₋₆ alkyl), —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆alkyl)C(═O)(C₁₋₆ alkyl), —NHCO₂(C₁₋₆ alkyl), —NHC(═O)N(C₁₋₆ alkyl)₂,—NHC(═O)NH(C₁₋₆ alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆alkyl), —OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆alkyl), —C(═NH)NH₂, —OC(═NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆ alkyl),—OC(NH)NH₂, —NHC(NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆ alkyl),—SO₂N(C₁₋₆ alkyl)₂, —SO₂NH(C₁₋₄ alkyl), —SO₂NH₂, —SO₂C₁₋₆ alkyl,—SO₂OC₁₋₆ alkyl, —OSO₂C₁₋₄ alkyl, —SOC₁₋₆ alkyl, —Si(C₁₋₆ alkyl)₃,—OSi(C₁₋₆ alkyl)₃-C(═S)N(C₁₋₆ alkyl)₂, C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂,—C(═O)S(C₁₋₆ alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)₂(C₁₋₆alkyl), —P(═O)(C₁₋₆ alkyl)₂, —OP(═O)(C₁₋₆ alkyl)₂, —OP(═O)(OC₁₋₆alkyl)₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, 5-10 memberedheteroaryl; or two geminal R^(gg) substituents can be joined to form ═Oor ═S;

wherein X⁻ is a counterion.

As used herein, the term “hydroxyl” or “hydroxy” refers to the group—OH. The term “substituted hydroxyl” or “substituted hydroxyl,” byextension, refers to a hydroxyl group wherein the oxygen atom directlyattached to the parent molecule is substituted with a group other thanhydrogen, and includes groups selected from —OR^(aa), —ON(R^(bb))₂,—OC(═O)SR^(aa), —OC(═O)R^(aa), —OCO₂R^(aa), —OC(═O)N(R^(bb))₂,—OC(═NR^(bb))R^(aa), —OC(═NR^(bb))OR^(aa), —OC(═NR^(bb))N(R^(bb))₂,—OS(═O)R^(aa), —OSO₂R^(aa), —OSi(R^(aa))₃, —OP(R^(cc))₂, —OP(R^(cc))₃,—OP(═O)₂R^(aa), —OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —OP(═O)₂N(R^(bb))₂,and —OP(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as definedherein.

As used herein, the term “thiol” or “thio” refers to the group —SH. Theterm “substituted thiol” or “substituted thio,” by extension, refers toa thiol group wherein the sulfur atom directly attached to the parentmolecule is substituted with a group other than hydrogen, and includesgroups selected from —SR^(aa), —S═SR^(cc), —SC(═S)SR^(aa),—SC(═O)SR^(aa), —SC(═O)OR^(aa), and —SC(═O)R^(aa), wherein R^(aa) andR^(cc) are as defined herein.

As used herein, the term, “amino” refers to the group —NH₂. The term“substituted amino,” by extension, refers to a monosubstituted amino ora disubstituted amino, or a trisubstituted amino, as defined herein. Incertain embodiments, the “substituted amino” is a monosubstituted aminoor a disubstituted amino group.

As used herein, the term “monosubstituted amino” refers to an aminogroup wherein the nitrogen atom directly attached to the parent moleculeis substituted with one hydrogen and one group other than hydrogen, andincludes groups selected from —NH(R^(bb)), —NHC(═O)R^(aa), —NHCO₂R^(aa),—NHC(═O)N(R^(bb))₂, —NHC(═NR^(bb))N(R^(bb))₂, —NHSO₂R^(aa),—NHP(═O)(OR^(cc))₂, and —NHP(═O)(NR^(bb))₂, wherein R^(aa), R^(bb) andR^(cc) are as defined herein, and wherein e of the group —NH(R^(bb)) isnot hydrogen.

As used herein, the term “disubstituted amino” refers to an amino groupwherein the nitrogen atom directly attached to the parent molecule issubstituted with two groups other than hydrogen, and includes groupsselected from —N(R^(bb))₂, —NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa),—NR^(bb)C(═O)N(R^(bb))₂, —NR^(bb)C(═NR^(bb))N(R^(bb))₂,—NR^(bb)SO₂R^(aa), —NR^(bb)P(═O)(OR^(cc))₂, and —NR^(bb)P(═O)(NR^(bb))₂,wherein R^(aa), R^(bb), and R^(cc) are as defined herein, with theproviso that the nitrogen atom directly attached to the parent moleculeis not substituted with hydrogen.

As used herein, the term “trisubstituted amino” refers to an amino groupwherein the nitrogen atom directly attached to the parent molecule issubstituted with three groups, and includes groups selected from—N(R^(bb))₃ and —N(R^(bb))₃ ⁺X⁻, wherein R^(bb) and X⁻ are as definedherein.

As used herein, the term “sulfonyl” refers to a group selected from—SO₂N(R^(bb))₂, —SO₂R^(aa), and —SO₂OR^(aa), wherein R^(aa) and R^(bb)are as defined herein.

As used herein, the term “sulfinyl” refers to the group —S(═O)R^(aa),wherein R^(aa) is as defined herein.

As used herein, the term “carbonyl” refers a group wherein the carbondirectly attached to the parent molecule is sp² hybridized, and issubstituted with an oxygen, nitrogen or sulfur atom, e.g., a groupselected from ketones (—C(═O)R^(aa)), carboxylic acids (—CO₂H),aldehydes (—CHO), esters (—CO₂R^(aa), —C(═O)SR^(aa), —C(═S)SR^(aa)),amides (—C(═O)N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa), —C(═S)N(R^(bb))₂), andimines (—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa)),—C(═NR^(bb))N(R^(bb))₂), wherein R^(aa) and R^(bb) are as definedherein.

As used herein, the term “silyl” refers to the group —Si(R^(aa))₃,wherein R^(aa) is as defined herein.

As used herein, a “counterion” is a negatively charged group associatedwith a positively charged quarternary amine in order to maintainelectronic neutrality. Exemplary counterions include halide ions (e.g.,F⁻, Cl⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻, HSO₄ ⁻, sulfonate ions(e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate,benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate,ethanoate, propanoate, benzoate, glycerate, lactate, tartrate,glycolate, and the like).

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quarternary nitrogenatoms. Exemplary nitrogen atom substitutents include, but are notlimited to, hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa),—C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc),—SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)S R^(cc), —C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂,—P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, or two R^(cc) groups attached to an N atom arejoined to form a 3-14 membered heterocyclyl or 5-14 membered heteroarylring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl,aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or5 R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc) and R^(dd) are asdefined above.

In certain embodiments, the substituent present on the nitrogen atom isan amino protecting group. Amino protecting groups include, but are notlimited to, —OH, —OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂,—CO₂R^(aa), —SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aralkyl, aryl, and heteroaryl is independently substitutedwith 0, 1, 2, 3, 4, or 5 R^(dd) groups, and wherein R^(aa), R^(bb),R^(cc) and R^(dd) are as defined herein. Amino protecting groups arewell known in the art and include those described in detail inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

These and other exemplary substituents are described in more detail inthe Detailed Description, the Examples and in the claims. The inventionis not intended to be limited in any manner by the above exemplarylisting of substituents.

The compounds useful in the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. As used herein, the term “isomers” includes anyand all geometric isomers and stereoisomers. For example, “isomers”include cis- and trans-isomers, E- and Z-isomers, R- and S-enantiomers,diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, andother mixtures thereof, as falling within the scope of the invention.For instance, an isomer/enantiomer may, in some embodiments, be providedsubstantially free of the corresponding enantiomer, and may also bereferred to as “optically enriched.” “Optically enriched,” as usedherein, means that the compound is made up of a significantly greaterproportion of one enantiomer. In certain embodiments the compound of thepresent invention is made up of at least about 90% by weight of aparticular enantiomer. In other embodiments the compound is made up ofat least about 95%, 98%, or 99% by weight of a particular enantiomer. Adesired enantiomer may be isolated from a racemic mixture by any methodknown to those skilled in the art, including chiral high pressure liquidchromatography (HPLC) or the formation and crystallization of chiralsalts, or the enantiomer may be prepared by asymmetric syntheses. See,for example, Jacques, et al., Enantiomers, Racemates and Resolutions(Wiley Interscience, New York, 1981); Wilen, S. H., et al., Tetrahedron33:2725 (1977); Eliel, E. L. Stereochemistry of Carbon Compounds(McGraw-Hill, N Y, 1962); Wilen, S. H. Tables of Resolving Agents andOptical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press,Notre Dame, Ind. 1972).

As used herein, the term “tautomer” includes two or moreinterconvertable compounds resulting from at least one formal migrationof a hydrogen atom and at least one change in valency (e.g., a singlebond to a double bond, a triple bond to a single bond, or vice versa).The exact ratio of the tautomers depends on several factors, includingtemperature, solvent, and pH. Tautomerizations (i.e., the reactionproviding a tautomeric pair) may catalyzed by acid or base. Exemplarytautomerizations include keto-to-enol; amide-to-imide; lactam-to-lactim;enamine-to-imine; and enamine-to-(a different) enamine tautomerizations.

The term “carbohydrate” refers to a sugar or polymer of sugars. Theterms “saccharide”, “polysaccharide”, “carbohydrate”, and“oligosaccharide”, may be used interchangeably. Most carbohydrates arealdehydes or ketones with many hydroxyl groups, usually one on eachcarbon atom of the molecule. Carbohydrates generally have the molecularformula C_(n)H_(2n)O_(n). A carbohydrate may be a monosaccharide, adisaccharide, trisaccharide, oligosaccharide, or polysaccharide. Themost basic carbohydrate is a monosaccharide, such as glucose, sucrose,galactose, mannose, ribose, arabinose, xylose, and fructose.Disaccharides are two joined monosaccharides. Exemplary disaccharidesinclude sucrose, maltose, cellobiose, and lactose. Typically, anoligosaccharide includes between three and six monosaccharide units(e.g., raffinose, stachyose), and polysaccharides include six or moremonosaccharide units. Exemplary polysaccharides include starch,glycogen, and cellulose. Carbohydrates may contain modified saccharideunits such as 2′-deoxyribose wherein a hydroxyl group is removed,2′-fluororibose wherein a hydroxyl group is replace with a fluorine, orN-acetylglucosamine, a nitrogen-containing form of glucose. (e.g.,2′-fluororibose, deoxyribose, and hexose). Carbohydrates may exist inmany different forms, for example, conformers, cyclic forms, acyclicforms, stereoisomers, tautomers, anomers, and isomers.

The phrase, “pharmaceutically acceptable derivative”, as used herein,denotes any pharmaceutically acceptable salt, ester, or salt of suchester, of such compound, or any other adduct or derivative which, uponadministration to a patient, is capable of providing (directly orindirectly) a compound as otherwise described herein, or a metabolite orresidue thereof. Pharmaceutically acceptable derivatives thus includeamong others pro-drugs.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, Berge et al.,describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds useful inthis invention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid ormalonic acid or by using other methods used in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Other pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium, quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate, and arylsulfonate.

Additionally, as used herein, the term “pharmaceutically acceptableester” refers to esters that hydrolyze in vivo and include those thatbreak down readily in the human body to leave the parent compound or asalt thereof. Suitable ester groups include, for example, those derivedfrom pharmaceutically acceptable aliphatic carboxylic acids,particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, inwhich each alkyl or alkenyl moeity advantageously has not more than 6carbon atoms. Examples of particular esters include formates, acetates,propionates, butyrates, acrylates and ethylsuccinates.

Furthermore, the term “pharmaceutically acceptable prodrugs” as usedherein refers to those prodrugs of the compounds useful in the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the issues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds useful in the invention. The term “prodrug” refers tocompounds that are rapidly transformed in vivo to yield the parentcompound of the above formula, for example by hydrolysis in blood. Apro-drug is a derivative of a compound, usually with significantlyreduced pharmacological activity, which contains an additional moiety,which is susceptible to removal in vivo yielding the parent molecule asthe pharmacologically active species. An example of a pro-drug is anester or an ether which is cleaved in vivo to yield a compound ofinterest. Pro-drugs of a variety of compounds, and materials and methodsfor derivatizing the parent compounds to create the pro-drugs, are knownand may be adapted to the present invention. The biological activity ofpro-drugs and pro-drugs may also be altered by appending a functionalityonto the compound, which may be catalyzed by an enzyme. Also, includedare oxidation and reduction reactions, including enzyme-catalyzedoxidation and reduction reactions. A thorough discussion of prodrugs isprovided in T. Higuchi and V. Stella, Pro-drugs as Novel DeliverySystems, Vol. 14 of the A.C.S. Symposium Series, and in Edward B. Roche,ed., Bioreversible Carriers in Drug Design, American PharmaceuticalAssociation and Pergamon Press, 1987, both of which are incorporatedherein by reference.

As used herein the term “inhibit” means to reduce the amount of kinaseactivity to a level or amount that is statistically significantly lessthan an initial level, which may be a baseline level of kinase activity.

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed. In other embodiments,treatment may be administered in the absence of symptoms. For example,treatment may be administered to a susceptible individual prior to theonset of symptoms (e.g., in light of a history of symptoms and/or inlight of genetic or other susceptibility factors). Treatment may also becontinued after symptoms have resolved, for example to prevent or delaytheir recurrence.

As used herein, unless otherwise specified, the terms “prevent,”“preventing” and “prevention” contemplate an action that occurs before asubject begins to suffer from the specified disease or disorder, whichinhibits or reduces the severity of the disease or disorder.

The terms “administer,” “administering,” or “administration,” as usedherein refers to implanting, absorbing, ingesting, injecting, orinhaling the compound.

The term “subject” refers to any animal. The subject may be at any stageof development. A “subject” to which administration is contemplatedincludes, but is not limited to, humans (i.e., a male or female of anyage group, e.g., a pediatric subject (e.g, infant, child, adolescent) oradult subject (e.g., young adult, middle-aged adult or senior adult))and/or other primates (e.g., cynomolgus monkeys, rhesus monkeys);mammals, including commercially relevant mammals such as cattle, pigs,horses, sheep, goats, cats, and/or dogs; and/or birds, includingcommercially relevant birds such as chickens, ducks, geese, and/orturkeys. In some embodiments, the subject is a rodent. In certainembodiments, the subject is an experimental animal such as a mouse, rat,dog, or non-human primate. In certain embodiments, the subject is atransgenic animal.

The term “proliferative disorder” as used herein refers to any diseaseassociated with an undesired and/or abnormal proliferation of cells. Thecells may be any type of cell found in the subject. The proliferationmay be due to any cause (e.g., any genetic mutation, any signal).

A therapeutically effective amount of a compound comprises administeringan amount necessary to achieve a desired result. The exact amountrequired will vary from subject to subject, depending on the species,age, general condition of the subject, the severity of the disease, theparticular anticancer agent, its mode of administration, the desiredoutcome, and the like. In certain embodiments of the present invention,a “therapeutically effective amount” of a compound or pharmaceuticalcomposition is that amount effective for inhibiting cell proliferationin a subject or a biological sample (e.g., in cells). In certainembodiments, cell proliferation is inhibited by about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, or about 99%. In certain embodiments, the compoundinhibits cell proliferation by at least about 25%, at least about 50%,at least about 75%, or at least about 90%. In certain embodiments of thepresent invention, a “therapeutically effective amount” refers to anamount of a compound or composition sufficient to inhibit cellproliferation, or refers to an amount of a compound or compositionsufficient to reduce the tumor burden in a subject. In certainembodiments, the tumor burden is reduced by about 1%, about 5%, about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, about 95%, or about 99%. In certain embodiments,the tumor burden is reduced by at least about 25%, at least about 50%,at least about 75%, or at least about 90%. In certain embodiments of thepresent invention a “therapeutically effective amount” of the compoundor pharmaceutical composition is that amount effective for reducing orinhibiting the growth of tumor cells and/or killing tumor cells.

As used herein, a “prophylactically effective amount” of a compound isan amount sufficient to prevent a disease or disorder, or one or moresymptoms associated with the disease or disorder, or prevent itsrecurrence. A prophylactically effective amount of a compound means anamount of therapeutic agent, alone or in combination with other agents,which provides a prophylactic benefit in the prevention of the diseaseor disorder. The term “prophylactically effective amount” can encompassan amount that improves overall prophylaxis or enhances the prophylacticefficacy of another prophylactic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Role of Cdc7 in the G1→S Phase Transition: Schematic diagram ofthe initiation of DNA replication in mammalian cells. The process can bedivided into several distinct phases: assembly, activation, andelongation. During assembly, the origin of DNA replication (ori) isbound by ORC1-6, the origin recognition complex that serves as aplatform for pre-replication complex (pre-RC) assembly during the G1phase of the cell cycle. This includes binding of the accessory proteinsCdc6, Cdt1, and the MCM2-7 protein complex that serves as thereplicative helicase. At the G1/S phase transition, the pre-RCs areactivated by both cyclin dependent kinase (CDK) and Cdc7 kinaseresulting in the establishment of the replication fork. Forkestablishment includes recruitment of single strand DNA binding protein(RPA) and the DNA polymerases required for leading and lagging strandsynthesis. Regulation of Cdc6 and Cdt1 via phosphorylation and proteincomplex assembly with geminin, respectively, inhibits further assemblyof functional pre-RCs insuring genomic integrity.

FIG. 2. Elutriation profile of PhALL3.1: Centrifugal elutriation wasused to synchronize the recently established Philadelphia chromosomepositive acute lymphoblastic leukemia cell line (PhALL3.1). Anasynchronous culture (4 liters) was pelleted, resuspended in a smallvolume (20 ml), and loaded into the elutriation rotor as outlined by themanufacturer (Beckman). After equilibration and washing, cell sampleswere eluted from the rotor based on size with the smaller cells exitingthe rotor first. Ten fractions (250 ml) were taken across the gradientand standard FACS analysis performed on 10 ml of each fraction lookingat DNA content. G1, S, and G2 phase cell populations are indicated inthe asynchronous profile. This method allows for isolation of phasespecific cell populations.

FIG. 3. Granaticin A Inhibits G1→S Transition: Granaticin A induces cellcycle arrest in G1 phase cells. Ph-ALL3.1 cells were subjected tostandard centrifugal elutriation conditions and the G1 phase fraction,as determined by FACS analysis (Time Zero), was released into granaticinA (1 mM) or vehicle control (DMSO) for the indicated times. Cell samplespost-release into granaticin A or vehicle control (DMSO) were pulsed for2 hours with ³H-thymidine and samples were collected, washed, and acidprecipitable counts were measured by scintillation counting. X-axis istime post-release into compound or control (in hours) and y-axis iscounts per minute (CPM). The graph demonstrates that while the controlcells progress through a normal S phase with a peak in incorporation asexpected from the cell cycle profile observed, granaticin A treatedsamples stop incorporating ³H-thymidine with cell cycle profiles showingG1 phase arrest. This implies that there is a complete block to enter Sphase (and activate origin firing) in the G1 population, correspondingto inhibition of Cdc7 kinase activity.

FIG. 4. Granaticin A induces Caspase 3-mediated apoptosis: 10 hoursfollowing release of G1 synchronized PhALL3.1 cells into either control(DMSO) or granaticin A, cell samples were taken and caspase 3 activitywas measured using a standard fluorometric assay as described in Gao etal., “Dimeric Smac/Diablo Peptide Directly Relieves Caspase-3 Inhibitionby XIAP” Journal Biological Chemistry (2007) 282:30718-30727.

FIG. 5. Granaticin A Induces Arrest and Apoptosis in S Phase cells:Granaticin A induces cell cycle arrest and apoptosis in S phase cells.Ph-ALL3.1 cells were subjected to standard centrifugal elutriationconditions and the S phase fraction, as determined by FACS analysis, wasreleased into granaticin A (1 μM) or vehicle control (DMSO) for theindicated times and FACS analysis was performed. Displayed are FACSprofiles with DNA content on the x-axis and cell number on the y-axisdemonstrating cell cycle progression in the control samples and arrestin the granaticin A treated samples with subsequent apoptosis as thesub-G1 fraction increases with time. Apoptosis was confirmed inadditional independent experiments. Asynchronous and time zerocorrespond to an asynchronous cell sample and the elutriated S phasefraction prior to release, respectively.

FIG. 6. Thymidine incorporation in S-phase: ³H-Thymidine incorporationduring the release experiment outlined in the slide above. Cell samplespost-release into granaticin A or vehicle control (DMSO) were pulsed for2 hours with ³H-thymidine, and samples were collected, washed, and acidprecipitable counts were measured by scintillation counting. X-axis istime post-release into compound or control (in hours) and y-axis iscounts per minute (CPM). The graph demonstrates that while the controlcells progress through a normal S phase with a peak in incorporation asexpected from the cell cycle profile observed, granaticin A treatedsamples display markedly reduced incorporation early and eventually stopincorporating ³H-thymidine with cell cycle profiles showing S phasearrest. This implies that not only is late origin firing inhibited, butthat replication fork progression is also inhibited by granaticin A.

FIG. 7. Granaticin A Inhibits Mcm2 phosphorylation: Followingsynchronization in S phase and release, PhALL3.1 cells from theexperiments of FIG. 5 and FIG. 6 were collected at the indicated timespost release into either control (DMSO) or granaticin A and extractswere subjected to Western blotting to detect total Mcm2 protein. Thisblot shows inhibition of Mcm2 phosphorylation as early as 2 hours postrelease into granaticin A and confirms target inhibition by granaticin Awithin the cell.

FIG. 8. Mcm2 phosphorylation by Cdc7-Phosphorylated forms of Mcm2migrate faster in SDS-PAGE gels. Using highly purified human recombinantCdc7 and Mcm2, standard in vitro kinase reactions were performed in thepresence of radioactive ATP and products were separated on SDS-PAGEgels, stained with silver, and autoradiography was performed to detectphosphorylated forms of Mcm2. Control is a negative control for kinaseactivity, DMSO is a positive control for kinase activity, granaticin Ais a small molecule inhibitor of Cdc7 kinase activity.

FIG. 9. Granaticin A Induces Cell Cycle Arrest in Hela S3 Cells: HeLa S3cells (cervical carcinoma) were synchronized at the G1/S transitionusing standard double thymidine block and release. Synchronized cells(time zero) were released into media containing granaticin A for theindicated times and standard FACS analysis performed looking at DNAcontent. This study shows that in HeLa cells S phase progression is alsoblocked by exposure to granaticin A.

FIG. 10. Granaticin B Induces Cell Cycle Arrest of Hela S3 Cells: Anidentical experiment to that described in FIG. 9 was performed usinggranaticin B and revealed similar results.

FIG. 11. Cell cycle arrest in Hela S3 cells: Morphologic appearance ofcontrol (DMSO), granaticin A, and granaticin B treated HeLa S3 cellsfollowing 15 hours release from double thymidine block. While controlcells look normal and healthy, granaticin A treated cells appeararrested with flattened morphology and peri-nuclear vacuolizationconsistent with induction of apoptosis. This phenotype is morepronounced in the granaticin B treated cells as one now sees nuclearblebbing and cell death. The corresponding FACS profiles are shown.

FIGS. 12-14. Inhibition of Granaticin A Induced Caspase-3 Activity inHeLa cells. Growth curves in the presence of the indicated micromolarconcentrations of granaticin A were performed and caspase-3 activity wasmeasured following addition of a substrate that when cleaved bycaspase-3 results in light emission which is quantitated by afluorimeter (y-axis) as a function of time (x-axis).

FIG. 12: HeLa cells alone;

FIG. 13: Bcl-XL overexpressing HeLa cells; and

FIG. 14: Bcl-XL overexpressing HeLa cells in the presence of 10micromolar Ac-DEVD-CHO (a caspase-3 peptide aldehyde inhibitor)demonstrating activation of caspase-3 and apoptosis in granaticin Atreated HeLa cells that is rescued by both overexpression of Bcl-XL andchemical inhibition of caspase-3 activity.

FIG. 15. Granaticin A Does Not Activate the S Phase Checkpoint in HeLaCells: Exposure of HeLa cells to granaticin A does not activate the Sphase checkpoint and results in increased double strand DNA breaks. HeLacells were incubated in the presence of granaticin A or carrier control(DMSO) for the indicated times and whole cell extracts were subjected toSDS-PAGE and western blot analysis. Asyn=asynchronously growing HeLacells. HU=HeLa cells exposed to hydroxyurea for the indicated time. HUwas used as a control for DNA damaging agent that is known to inhibitthe DNA damage checkpoint response. Again, we observed that Mcm2mobility is shifted in the cells treated with granaticin A. MCM2mobility is not affected by HU. Activation of the DNA damage/replicationcheckpoint correlates with ATM/ATR dependent phosphorylation of Chk1 atSer345. ChK1 Ser345 levels are below detection limits, suggesting thatwith Cdc7 inhibition, a DNA replication checkpoint is either notefficiently activated or maintained and that inhibitory signalspreventing cells from progressing in the cell cycle might not begenerated.

FIGS. 16A-16B. Granaticin A and Granaticin B inhibit multiple tumortypes: The indicated cell lines and primary patient samples were testedagainst the compounds listed in standard cytotoxicity assays (12 pointdose response using Alamar blue reduction as an indicator of cellviability). Assays were done in triplicate and 1050 determinations wererun in 12-point serial dilutions from 10 mM to 5 nM. 1050 values aredisplayed with darker shades indicating low nanomolar values and lightershades high micromolar values, respectively. FIGS. 16A and 16B summarizethe IC50 determinations for granaticin A and granaticin B, as well asDerivatives 1 and 2 (FIG. 16A) against multiple non-small cell lungcancer cell lines, ovarian cancer cell lines, mesothelioma, sarcoma, andmelanoma cell lines including cells isolated from primary patient ocularmelanoma samples (124859-A and -SB). These include ras and EGFRgatekeeper mutation containing cell lines in addition to p53 mutant celllines and other known chemotherapy resistant cell lines. Specifically,this included multi-drug resistance phenotype (MDR-overexpressing) celllines from multiple hematologic and solid tumor cell lines. Table 2provided herein summarizes the data provided in FIG. 16B (see Examples).Table 1 highlights a series of primary patient leukemia samples-bothacute and chronic and treatment naïve and refractory tested againstgranaticins A and B and Derivatives 1 and 2 (see Examples). FIGS. 16Aand 16B, and Tables 1-2, together, show that granaticin A, Derivatives 1and 2, and especially granaticin B, are pan-active against multipletumor cell lines and in primary patient samples from patients withchronic and acute leukemias both treatment naïve and chemotherapyrefractory.

FIG. 17. t(9:22)(q34;q11) Acute Lymphocytic Leukemia cell line: Based onthe results of the cytotoxicity assays, granaticin A and granaticin Bwere chosen to pursue studies in mice using the Ph-ALL3.1 mouse model.The PhALL3.1 cell line was retrovirally transduced with a GFP-fireflyluciferase expressing construct using standard techniques. The infectedcells were sorted by FACS for CD19 and GFP co-expression. Panel A is theFACS profile of the cells before transduction and Panel B is the FACSprofile of the cells after transduction demonstrating CD19-GFP doublepositive cells which were then used to inject immunocompromisedSCID-Beige mice. Ph+ ALL cells were isolated from the pleural fluid of apatient treated on the Leukemia Service, and used to create a cell linethat grows readily in culture. The cytogenetics were confirmed bykaryotype, FISH, RT-PCR, and Western blot analysis for p190 bcr-abl. Thecell line was then transduced with a retrovirus GFP-firefly luciferaseconstruct. Granaticin A has been shown to be very active against thiscell line in the cell based assays.

FIGS. 18A-18B. Mouse survival following tumor cell inoculation: 5million PhALL3.1 tumor cells were injected into the tail veins of acohort of eight SCID-Beige immunocompromised mice and diseaseprogression was monitored on the indicated days using in vivobioluminescence imaging. FIG. 18A shows that this mouse model of humanacute lymphoblastic leukemia faithfully duplicates the progression seenin human disease. FIG. 18B shows that when left untreated these micemeet criteria for euthanasia by day 41 indicating a rapid, progressive,and lethal leukemia.

FIGS. 19A-19B. Granaticin A Inhibits growth of PhALL3.1 in vivo: Using aSCID-Beige mouse intraperitoneal tumor model of PhALL3.1, mice wereintraperitoneally treated with vehicle control (DMSO) or the indicateddoses of granaticin A on days 3, 6, 9, and 12. Day 16 in vivobioluminescence imaging (FIG. 19A) and quantitation of tumor volume areshown (FIG. 19B) and indicate 90% reduction in tumor volume by Day 16 inmice treated at the highest dose (3.0 mg/kg). Separate necropsy datarevealed no significant (grade 2 or higher) organ toxicity in all miceexamined at each dose level.

FIGS. 20A-20C. Granaticin B Inhibits Ph-ALL3.1 growth in vivo: Given therapid intravenous clearance of granaticin A by the liver, granaticin Bwas used in a continuous infusion experiment with the PhALL3.1 animalmodel. Diffusion pumps containing granaticin B were surgically implantedin the flanks of 5 cohorts of mice on day +3 after injection of 5million cells via the tail veins, and the drug (or control-C-DMSO) atthe indicated concentration was allowed to diffuse for seven days beforepump removal. The mice were imaged on day 12 (FIGS. 20A-20B) and tumorvolume remaining was calculated (FIG. 20C). 95% tumor reduction was seenat the highest dose. The mice tolerated the procedure without obviousorgan toxicity as seen on necropsy and indicate that granaticin B isstable enough when given intravenously to achieve efficacy against thislethal tumor model.

FIG. 21. Granaticin B stability study comparison in HP/MP: Standardplasma stability studies were conducted with Granaticin B. The recoveryof Granaticin B was measured by mass spectrometry.

FIG. 22A-22C. Granaticin B is Effective Against Multiple Solid Tumors:granaticin B is efficacious against mouse models of melanoma (FIG. 22A),non-small cell lung cancer (FIG. 22B), and ovarian cancer (FIG. 22C).For the ovarian cancer experiments, SCID-Beige mice were injected with 5million OVCAR3 cells that had been transduced with a GFP-fireflyluciferase expressing plasmid (Frattini, M. G. and Brentjens, R. J.,unpublished data). These cells were then injected intraperitoneally into4 cohorts of mice on Day 1. From days 7-14 the mice were injected dailywith the indicated concentrations of granaticin B in the intraperitonealspace. Imaging (luciferase activity) was acquired on day 42 and revealedessentially no disease in the mice treated at 3 mg/kg granaticin B. Forthe melanoma and non small-cell lung cancer models, SKMEL-28 and A459cells, respectively were used in standard nude mouse xenograftexperiments. Nude mice were treated with 2.5 mg/kg/day granaticin B in acontinuous fashion using osmotic diffusion pumps. Sections of thetreated xenografts are shown after euthanasia at day 28 and H&Estaining. Control sections revealed no evidence of necrosis or celldeath (not shown).

FIG. 23A-23C. Granaticin A and B are not cytotoxic to normal diploidcells: FIGS. 23A-23B: Granaticin B exposure is not cytotoxic to normaldiploid cells. Either HeLa or RPE (retinal pigment epithelial cells, anormal human diploid cell line) cells were incubated in carrier control(DMSO) or granaticin B (1 microM) for 24 hours and samples weresubjected to standard FACS analysis with the x-axis indicating DNAcontent (FIG. 23A). The cell cycle profile for the RPE cells isessentially unaffected by granaticin B exposure where the HeLa cellcycle profile indicates apoptotic cell death with an increase in sub-G1DNA containing cells and loss of normal cell cycle progression. Extractsfrom cells at the indicated times, exposed to either DMSO or granaticinB (1 microM), were used in standard caspase-3 activity assays to monitorfor induction of apoptosis and show that capsae-3 is only activated inHeLa cells exposed to granaticin B (FIG. 23B). Granaticin B exposuredoes not result in caspase-3 activation in RPE cells. FIG. 23C:Granaticin A exposure is not cytotoxic to normal diploid cells. EitherHeLa or RPE (retinal pigment epithelial cells, a normal diploid cellline) cells were incubated in carrier control (DMSO) or granaticin A for24 hours and samples were subjected to standard FACS analysis with FL2indicating DNA content. The cell cycle profile for the RPE cells isunaffected by granaticin A exposure where the HeLa cell cycle profileindicates apoptotic cell death with an increase in sub-G1 DNA containingcells.

FIGS. 24A-24B. Granaticin B is very effective against JOKE-2 cells. TheJOKE-2 cell line was derived from a patient with Philadelphia chromosomepositive Acute Lymphoblastic Leukemia (Ph-ALL). The patient wasclinically resistant to imatinib mesylate and the Bcr-Abl kinase wasfound to have the following high-risk resistance mutations in the Ablkinase domain (F317L, F359V, T3151, and E255K). FIG. 24A: Joke-2 cellswere grown in the presence of DMSO control or granaticin B (1 microM)for the indicated times and viability was measured after staining a cellsample in trypan blue (Sigma-Aldrich). FIG. 24B: Extracts from cells atthe indicated times, exposed to either DMSO or granaticin B (1 microM),were used in standard caspase-3 activity assays to monitor for inductionof apoptosis and show that capsae-3 is only activated in cells exposedto granaticin B.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

The current invention derives from the discovery that thebenzoisochromane quinones (BIQs), such as the granaticins, inhibitprotein kinase activity. The granaticins, in particular, were found toinhibit Cdc kinase activity based upon hits identified fromhigh-thoroughput screening (HTS) of over 300,000 compounds for theirability to inhibit a heterodimer of a kinase (Cdc7) and an activator(Dbf4) that phosphorylates serine and threonine residues.

The present invention provides methods for the inhibition of proteinkinase activity, such as Cdc kinase activity, and particularly, Cdc7kinase activity. The inhibitory compounds useful in the methods include,but are not limited to, granaticin A, granaticin B, dihydrogranaticin A,dihydrogranaticin B, medermycin, actinorhodin, benzoisochromanequinones,as shown herein. The methods of the present invention are useful in thetreatment of kinase-related diseases or disorders. Specifically, themethods are useful in the treatment of proliferative disorders, such ascancer, benign tumors, inflammatory disorders, and complicationsthereof. The present invention also provides pharmaceutical compositionsand kits for the treatment of various diseases.

Compounds Useful in the Invention

The present invention utilizes compounds of the Formula (A):

or pharmaceutically acceptable salts thereof,wherein:

each instance of R¹ and R⁴ is independently selected from the groupconsisting of hydrogen, carbonyl, silyl, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl;

each instance of R² and R³ is independently selected from the groupconsisting of hydrogen, halogen, —OH, substituted hydroxyl, —SH,substituted thiol, —NH₂, substituted amino, —CN, —NO₂, carbonyl, silyl,sulfinyl, sulfonyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, and optionally substituted heteroaryl; or R² and R³ are joined toform an optionally substituted carbocyclyl, optionally substitutedheterocyclyl, optionally substituted aryl, or optionally substitutedheteroaryl group;

R⁵ is hydrogen and R⁶ is selected from the group consisting of hydrogen,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl; or R⁵ and R⁶ are joined to form a direct bond;and

R⁷ is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, and optionally substituted heteroaryl.

In certain embodiments, R¹ is hydrogen. In certain embodiments, R¹ iscarbonyl, silyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl.

In certain embodiments, R⁴ is hydrogen. In certain embodiments, R⁴ isoptionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, or optionally substituted heteroaryl.

In certain embodiments, both R¹ and R⁴ are hydrogen, e.g., to provide acompound of the Formula (A-1):

or a pharmaceutically acceptable salt thereof,wherein R³, R⁴, R⁵, R⁶, and R⁷ are as defined herein.

In certain embodiments, R² is hydrogen. In certain embodiments, R² isoptionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl. In certain embodiments, R² is optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, or optionally substituted heteroaryl. In certainembodiments, R² is optionally substituted heterocyclyl (e.g., optionallysubstituted tetrahydropyranyl).

In certain embodiments, R² is an optionally substitutedtetrahydropyranyl group of the formula (i):

wherein:

each instance of R⁸ is independently selected from optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, —OH, substituted hydroxyl, —SH, substitutedthiol, —NH₂, substituted amino, —CN, —NO₂, —N₂, carbonyl, silyl,sulfonyl, and sulfinyl; and

m is 0 or an integer of between 1 and 5, inclusive.

In certain embodiments, each instance of R⁸ is independently selectedfrom optionally substituted alkyl, —OH, substituted hydroxyl, —SH,substituted thiol, —NH₂, substituted amino, carbonyl, and silyl. Incertain embodiments, each instance of R⁷ is independently selected fromoptionally substituted alkyl, —OH, substituted hydroxyl, —NH₂, andsubstituted amino. In certain embodiments, each instance of R⁸ isindependently selected from optionally substituted alkyl, —OH, andsubstituted amino (e.g., disubstituted amino). In certain embodiments,each instance of R⁸ is independently selected from —CH₃, —OH, and—N(CH₃)₂.

In certain embodiments, m is an integer of between 1 and 4, inclusive.In certain embodiments, m is an integer of between 1 and 3, inclusive.In certain embodiments, m is an integer of between 1 and 2, inclusive.In certain embodiments, m is 3.

In certain embodiments, each instance of R⁸ is independently selectedfrom —CH₃, —OH, and —N(CH₃)₂, and m is an integer of between 1 and 3,inclusive. For example, in this instance, in certain embodiments, R² isa substituted tetrahydropyranyl group of the formula (ii):

In certain embodiments, the substituted tetrahydropyranyl group of theformula (ii) is of the formula (iii):

Alternatively, in certain embodiments, R² is an optionally substitutedaryl or optionally substituted heteroaryl. In certain embodiments, R² isan optionally substituted aryl (e.g., a benzoisochromanequinone).

In certain embodiments R² is substituted benzoisochromanequinone of theformula (iv):

wherein R¹, R³, R⁴, R⁵, R⁶, and R⁷ are as defined herein.

In certain embodiments, the substituted benzoisochromanequinone offormula (iv) is of the formula (v):

Alternatively, in certain embodiments, R² and R³ are joined to form anoptionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, or optionally substituted heteroaryl group.In certain embodiments, R² and R³ are joined to form an optionallysubstituted carbocyclyl or optionally substituted heterocyclyl group. Incertain embodiments, R² and R³ are joined to form an optionallysubstituted heterocyclyl group (e.g., an optionally substituted2-oxabicyclo[2.2.2]octenyl group).

In certain embodiments, R² and R³ joined to form a substituted2-oxabicyclo[2.2.2]octenyl group of the formula (vi):

wherein each instance of R⁹ and R¹⁰ is independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, carbonyl, silyl, sulfonyl, andsulfinyl.

In certain embodiments, R⁹ is hydrogen. In certain embodiments, R⁹ isoptionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl. In certain embodiments, R⁹ is optionallysubstituted carbocyclyl or optionally substituted heterocyclyl. Incertain embodiments, R⁹ is optionally substituted heterocyclyl (e.g.,optionally substituted tetrahydropyranyl).

In certain embodiments, R⁹ is optionally substituted tetrahydropyranylgroup of the formula (vii):

wherein:

each instance of R¹¹ is independently selected from optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, —OH, substituted hydroxyl, —SH, substitutedthiol, —NH₂, substituted amino, —CN, —NO₂, —N₂, carbonyl, silyl,sulfonyl, and sulfinyl; and

p is 0 or an integer of between 1 and 5, inclusive.

In this instance, in certain embodiments, the R² and R³ are joined toform a substituted 2-oxabicyclo[2.2.2]octenyl group of the formula(viii):

In certain embodiments, each instance of R¹¹ is independently selectedfrom optionally substituted alkyl, —OH, substituted hydroxyl, —SH,substituted thiol, —NH₂, substituted amino, carbonyl, and silyl. Incertain embodiments, each instance of R¹⁰ is independently selected fromoptionally substituted alkyl, —OH, and substituted hydroxyl. In certainembodiments, each instance of R¹¹ is independently selected fromoptionally substituted alkyl and —OH. In certain embodiments, eachinstance of R¹¹ is independently selected from —CH₃ and —OH.

In certain embodiments, p is an integer of between 1 and 4, inclusive.In certain embodiments, p is an integer of between 1 and 3, inclusive.In certain embodiments, p is an integer of between 1 and 2, inclusive.In certain embodiments, p is 2.

In certain embodiments, each instance of R¹¹ is independently selectedfrom —CH₃ and —OH, and p is an integer of between 1 and 2, inclusive.For example, in this instance, in certain embodiments, R¹¹ is asubstituted tetrahydropyranyl group of the formula (ix):

In certain embodiments, the substituted tetrahydropyranyl group of theformula (ix) is of the formula (x):

In this instance, in certain embodiments, the R² and R³ are joined toform a substituted 2-oxabicyclo[2.2.2]octenyl group of the formula (xi):

In certain embodiments, R¹⁰ is optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, carbonyl, silyl,sulfonyl, or sulfinyl.

However, in certain embodiments, R¹⁰ is hydrogen. In this instance, incertain embodiments, the R² and R³ are joined to form a substituted2-oxabicyclo[2.2.2]octenyl group of the formula (xii):

In certain embodiments, both R⁹ and R¹⁰ are hydrogen. In this instance,in certain embodiments, the R² and R³ are joined to form a substituted2-oxabicyclo[2.2.2]octenyl group of the formula (xiii):

In certain embodiments, R⁵ is hydrogen and R⁶ is selected from the groupconsisting of selected from hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl.

In certain embodiments, both R⁵ and R⁶ are hydrogen. However, in certainembodiments, R⁵ is hydrogen and R⁶ is optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl.

Alternatively, in certain embodiments, R⁵ and R⁶ are joined to form adirect bond, e.g., to provide a dihydrofuran-2-one of the formula (xiv):

In certain embodiments, the dihydrofuran-2-one of the formula (xiv) isof the formula (xv):

In certain embodiments, the dihydrofuran-2-one of the formula (xiv) isof the formula (xvi):

In certain embodiments, R⁷ is hydrogen. In certain embodiments, R⁷ isoptionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, and optionallysubstituted heteroaryl. In certain embodiments, R⁷ is optionallysubstituted alkyl (e.g., —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, and thelike). In certain embodiments, R⁷ is —CH₃.

In certain embodiments, wherein R⁵ and R⁶ are joined to form a directbond, i.e., to provide a dihydrofuran-2-one of the formula (xiv), thecompound of Formula (A) is of the Formula (A-2):

or a pharmaceutically acceptable salt thereof. In certain embodiments,R⁷ is —CH₃. In certain embodiments, R¹ and R⁴ are hydrogen.

In certain embodiments, wherein R⁵ and R⁶ are joined to form adihydrofuran-2-one of the formula (xv), the compound of Formula (A-2) isof the Formula (A-3):

or a pharmaceutically acceptable salt thereof. In certain embodiments,R⁷ is —CH₃. In certain embodiments, R¹ and R⁴ are hydrogen.

In certain embodiments, wherein R⁵ and R⁶ are joined to form adihydrofuran-2-one of the formula (xvi), the compound of Formula (A-2)is of the Formula (A-4):

or a pharmaceutically acceptable salt thereof. In certain embodiments,R⁷ is —CH₃. In certain embodiments, R¹ and R⁴ are hydrogen.

In certain embodiments, wherein R¹ and R⁴ are hydrogen, the compound ofFormula (A-3) is of the Formula (A-5):

or a pharmaceutically acceptable salt thereof. In certain embodiments,R⁷ is —CH₃.

In certain embodiments, wherein R¹ and R⁴ are hydrogen, the compound ofFormula (A-4) is of the Formula (A-6):

or a pharmaceutically acceptable salt thereof. In certain embodiments,R⁷ is —CH₃.

In certain embodiments, wherein R² and R³ are joined to form asubstituted oxabicyclo[2.2.2]octenyl group of the formula (vi), thecompound of Formula (A-5) is of the Formula (A-7):

or a pharmaceutically acceptable salt thereof. In certain embodiments,R⁷ is —CH₃.

Exemplary compounds of the Formula (A) include, but are not limited to:

or pharmaceutically acceptable salts thereof.

The present invention also utilizes compounds of the Formula (B):

or pharmaceutically acceptable salts thereof,

wherein:

R¹² is hydrogen, carbonyl, silyl, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl;

each instance of R¹³ and R¹⁴ is independently selected from the groupconsisting of hydrogen, halogen, —OH, substituted hydroxyl, —SH,substituted thiol, —NH₂, substituted amino, —CN, —NO₂, carbonyl, silyl,sulfinyl, sulfonyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, and optionally substituted heteroaryl; or R¹³ and R¹⁴ are joinedto form an optionally substituted carbocyclyl, optionally substitutedheterocyclyl, optionally substituted aryl, or optionally substitutedheteroaryl group;

R⁵ is hydrogen and R⁶ is selected from the group consisting of hydrogen,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl; or R⁵ and R⁶ are joined to form a direct bond;and

R⁷ is hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl, and optionally substituted heteroaryl.

In certain embodiments, R¹² is hydrogen. In certain embodiments, R¹² iscarbonyl, silyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl.

In certain embodiments, R¹³ is hydrogen. In certain embodiments, R¹³ ishalogen, —OH, substituted hydroxyl, —SH, substituted thiol, —NH₂,substituted amino, —CN, —NO₂, carbonyl, silyl, sulfinyl, sulfonyl,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, and optionallysubstituted heteroaryl. In certain embodiments, R¹³ is —OH, substitutedhydroxyl, —SH, substituted thiol, —NH₂, substituted amino. In certainembodiments, R¹³ is —OH or substituted hydroxyl. In certain embodiments,R¹³ is —OH.

In certain embodiments, R¹⁴ is hydrogen. In certain embodiments, R¹⁴ isoptionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl. In certain embodiments, R¹⁴ is optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, or optionally substituted heteroaryl. In certainembodiments, R¹⁴ is optionally substituted heterocyclyl (e.g.,optionally substituted tetrahydropyranyl).

In certain embodiments, R¹⁴ is an optionally substitutedtetrahydropyranyl group of the formula (xvii):

wherein:

each instance of R¹⁵ is independently selected from optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, —OH, substituted hydroxyl, —SH, substitutedthiol, —NH₂, substituted amino, —CN, —NO₂, carbonyl, silyl, sulfonyl,and sulfinyl; and

q is 0 or an integer of between 1 and 5, inclusive.

In certain embodiments, each instance of R¹⁵ is independently selectedfrom optionally substituted alkyl, —OH, substituted hydroxyl, —SH,substituted thiol, —NH₂, substituted amino, carbonyl, and silyl. Incertain embodiments, each instance of R¹⁵ is independently selected fromoptionally substituted alkyl, —OH, substituted hydroxyl, —NH₂, andsubstituted amino. In certain embodiments, each instance of R¹⁵ isindependently selected from optionally substituted alkyl, —OH, andsubstituted hydroxyl. In certain embodiments, each instance of R¹⁵ isindependently selected from —CH₃ and —OH.

In certain embodiments, q is an integer of between 1 and 4, inclusive.In certain embodiments, q is an integer of between 1 and 3, inclusive.In certain embodiments, q is an integer of between 1 and 2, inclusive.In certain embodiments, q is 3.

In certain embodiments, each instance of R¹⁵ is independently selectedfrom —CH₃, —OH, and m is an integer of between 1 and 3, inclusive. Forexample, in this instance, in certain embodiments, R¹⁵ is a substitutedtetrahydropyranyl group of the formula (xviii):

Alternatively, in certain embodiments, R¹³ and R¹⁴ are joined to form anoptionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, or optionally substituted heteroaryl group.In certain embodiments, R¹³ and R¹⁴ are joined to form an optionallysubstituted carbocyclyl or optionally substituted heterocyclyl group. Incertain embodiments, R¹³ and R¹⁴ are joined to form an optionallysubstituted heterocyclyl group (e.g., an optionally substituted2-oxabicyclo[2.2.2]octenyl group).

In certain embodiments, R¹³ and R¹⁴ are joined to form a substituted2-oxabicyclo[2.2.2]octenyl group of the formula (xix):

wherein each instance of R¹⁶ and R¹⁷ is independently selected fromhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, carbonyl, silyl, sulfonyl, andsulfinyl.

In certain embodiments, R¹⁶ is optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, or optionally substituted heteroaryl. In certainembodiments, R¹⁶ is optionally substituted carbocyclyl or optionallysubstituted heterocyclyl. However, in certain embodiments, R¹⁶ ishydrogen, e.g., and R¹³ and R¹⁴ are joined to form a substituted2-oxabicyclo[2.2.2]octenyl group of the formula (XX):

In certain embodiments, R¹⁷ is optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, or optionally substituted heteroaryl. In certainembodiments, R¹⁷ is optionally substituted carbocyclyl or optionallysubstituted heterocyclyl. However, in certain embodiments, R¹⁷ ishydrogen, e.g., and R¹³ and R¹⁴ are joined to form a substituted2-oxabicyclo[2.2.2]octenyl group of the formula (xxi):

In certain embodiments, both R¹⁶ and R¹⁷ are hydrogen. In this instance,in certain embodiments, the R¹³ and R¹⁴ are joined to form a substituted2-oxabicyclo[2.2.2]octenyl group of the formula (xxii):

In certain embodiments, R⁵ is hydrogen and R⁶ is selected from the groupconsisting of selected from hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl.

In certain embodiments, both R⁵ and R⁶ are hydrogen. However, in certainembodiments, R⁵ is hydrogen and R⁶ is optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl.

Alternatively, in certain embodiments, R⁵ and R⁶ are joined to form adirect bond, e.g., to provide a dihydrofuran-2-one of the formula (xiv):

In certain embodiments, the dihydrofuran-2-one of the formula (xiv) isof the formula (xv):

In certain embodiments, the dihydrofuran-2-one of the formula (xiv) isof the formula (xvi):

In certain embodiments, R⁷ is hydrogen. In certain embodiments, R⁷ isoptionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, and optionallysubstituted heteroaryl. In certain embodiments, R⁷ is optionallysubstituted alkyl (e.g., —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, and thelike). In certain embodiments, R⁷ is —CH₃.

In certain embodiments, wherein R¹² is hydrogen, the compound of Formula(B) is of the Formula (B-1):

or a pharmaceutically acceptable salt thereof. In certain embodiments,R⁷ is —CH₃.

In certain embodiments, wherein R⁵ and R⁶ are joined to form a directbond, the compound of Formula (B-1) is of the Formula (B-2):

or a pharmaceutically acceptable salt thereof. In certain embodiments,R⁷ is —CH₃.

In certain embodiments, wherein R¹³ and R¹⁴ are joined to form asubstituted 2-oxabicyclo[2.2.2]octenyl group of the formula (xix), thecompound of Formula (B-1) is of the Formula (B-3):

or a pharmaceutically acceptable salt thereof. In certain embodiments,R⁷ is —CH₃.

In certain embodiments, wherein R¹³ is —OH and R¹⁴ is an optionallysubstituted tetrahydropyranyl group of the formula (xvii), the compoundof Formula (B-1) is of the Formula (B-4):

or a pharmaceutically acceptable salt thereof. In certain embodiments,R⁷ is —CH₃.

Exemplary compounds of the Formula (B) include, but are not limited to:

or pharmaceutically acceptable salts thereof.

In certain embodiments, the compounds of Formula (A) and (B) have anIC₅₀ of less than approximately 100 μM, e.g., less than approximately 10μM, e.g., less than approximately 1 μM, e.g., less than approximately0.1 μM, or e.g., less than approximately 0.01 μM.

The compounds described herein may be useful in methods, pharmaceuticalcompositions, and kits, for example, in the treatment and prevention ofproliferative disorders, such as cancer, benign tumors, inflammatorydisorders, and complications thereof.

Protein Kinases, Cdc7, and Dbf4

Compounds useful in the present invention are protein kinase inhibitors.The protein kinases constitute a group of enzymes that catalyse thephosphorylation of hydroxy groups of specific protein residues such astyrosine, serine or threonine residues. Said phosphorylations canextensively modify the function of proteins; thus, the protein kinasesplay an important role in the regulation of a great variety of cellularprocesses, notably including metabolism, cellular proliferation, celldifferentiation or cell survival. Among the various cellular functionsin which the activity of a protein kinase is involved, certain processesrepresent attractive targets for treating certain diseases. As anexample, we may notably mention angiogenesis and control of the cellcycle, in which the protein kinases can play an essential role. Theseprocesses are essential for the growth of solid tumours as well as forthe development of other diseases.

The protein kinases participate in signalling events that control theactivation, growth and differentiation of cells in response either toextracellular mediators, or to changes in the environment. In general,these kinases belong to two groups: those which phosphorylate serineand/or threonine residues preferentially, and those which phosphorylatetyrosine residues preferentially (S. K. Hanks and T. Hunter, FASEB. J.,1995, 9, pages 576-596). The serine/threonine kinases, for example, areisoforms of protein kinases C (A. C. Newton, J. Biol. Chem., 1995, 270,pages 28495-28498) and a group of cyclin-dependent kinases, such as Cdc2(Cdk1) (J. Pines, Trends in Biochemical Sciences, 1995, 18, pages195-197). The tyrosine kinases include growth factor receptors, such asthe epidermal growth factor (EGF) receptor (S. Iwashita and M.Kobayashi, Cellular Signalling, 1992, 4, pages 123-132), and cytosolickinases such as p56tck, p59fYn, ZAP-70 and the csk kinases (C. Chan etal., Ann. Rev. Immunol., 1994, 12, pages 555-592).

Abnormally high levels of protein kinase activity have been associatedwith many diseases, resulting in abnormal cellular functions. This canoccur either directly or indirectly, from a malfunction in the controlmechanisms of kinase activity, connected for example with a mutation,overexpression or inappropriate activation of the enzyme, or withoverproduction or underproduction of cytokines or of growth factors,which are also involved in signal transduction upstream or downstream ofthe kinases. In all these cases, selective inhibition of the action ofthe kinases offers hope of a beneficial effect. Among these proteinkinases, we may mention quite particularly protein kinase Cdc7. Cdc7 isa serine/threonine kinase that has been characterized at the molecularlevel as a factor that is essential for initiating DNA replication.

The catalytic activity of Cdc7, which is conserved throughout theeukaryotes, is dependent on its Dbf4 regulatory subunit. Although theexpression of Cdc7 (at the level of messenger and protein RNA) isconstant throughout the cell cycle, the level of expression of Dbf4 isin contrast dependent on the cell cycle, which induces an increase inCdc7 kinase activity during the G1-S transition. For this reason, Cdc7is given the designation DDK (Dbf-4-dependent kinase).

The principal activity of the Cdc7/Dbf4 complex occurs on initiation ofDNA replication during the S phase. It phosphorylates MCM2 which thusactivates the MCM (Mini-Chromosome Maintenance) complex, which is anessential component of DNA-helicase activity.

Cdc7 also plays an important role in mutagenesis, mainly induced byaction at the level of the DNA-damage pathways and checkpoints, inparticular at the ATR-dependent checkpoint, which prevents theinitiation of DNA replication in response to damage of thesingle-stranded type caused by chemical agents such as etoposide.

Cdc7 and Dbf4 are overexpressed in human tumour cell lines and in manytumour samples (lung, breast, thyroid, colon-rectum, oesophagus, uterus,testicle, liver) in comparison with the corresponding normal tissues(Hess, G. F., Drong, R. F., et al. 1998).

Experiments in suppressing Cdc7 expression using RNA interference (RNAi)technology show that inhibition of Cdc7 expression induces arrest of thecell cycle and prevents cellular proliferation of the human tumour celllines HeLa and HCT116, but has a limited effect on normal cells (normalhuman skin fibroblasts). This is reflected in a prolonged stoppage in G1that induces apoptosis in cells lacking p53 (>50% of tumours) but isreversible in normal cells [A. Montagnoli et al., CANCER RESEARCH 64,7110-7116, Oct. 1, 2004].

The inhibitors of Cdc7 kinase activity can constitute a novel categoryof targeted cytotoxic therapy as well as of inhibitors of DNAreplication. Such inhibitors would inhibit replication before thereplication forks are established, thus blocking replication withoutdamaging the DNA. The present application thus relates in particular tonovel inhibitors of protein kinase Cdc7 that can be used notably for thetreatment of abnormal cellular proliferation and more particularly inoncology.

Methods of Treatment

The methods involve uses of a compound of Formula (A) or (B), orpharmaceutically acceptable salt or pharmaceutical composition thereof.

In one aspect, the present invention provides a compound of Formula (A)or (B), or a pharmaceutically acceptable salt thereof, for use in thetreatment or prevention of a proliferative disorder.

In another aspect, the present invention provides use of a compound ofFormula (A) or (B), or a pharmaceutically acceptable salt thereof, forthe manufacture of a medicament for treating or preventing aproliferative disorder.

In yet another aspect, the present invention provides a method fortreating or preventing a proliferative disorder in a subject in needthereof, wherein the subject is administered a therapeutically effectiveamount of a compound of the Formula (A) or (B), or a pharmaceuticallyacceptable salt thereof.

In certain embodiments, the subject is human.

In certain embodiments, the compound induces apoptosis. In certainembodiments, compound induces S-phase arrest.

In certain embodiments, the compound is an inhibitor of a proteinkinase; and wherein the inhibition of a protein kinase is useful for thetreatment or prevention of the proliferative disorder. In certainembodiments, the protein kinase is Cdc7 kinase or the Dbf4 regulatorysubunit of Cdc7 kinase, and wherein the inhibition of Cdc7 kinase or theDbf4 regulatory subunit of Cdc7 kinase is useful for the treatment orprevention of the proliferative disorder. In some embodiments, theprotein kinase inhibitor is a Cdc7 kinase inhibitor or the Dbf4regulatory subunit of Cdc7 kinase.

In certain embodiments, the proliferative disorder is selected from thegroup consisting of cancer, myeloproliferative disorders (e.g.,polycythemia vera, essential thrombocytosis, meylofibrosis, and chronicmyelogenous leukemia (CML)), benign prostate hyperplasia, familialadenomatosis, polyposis, neuro-fibromatosis, psoriasis, vascular smoothcell proliferation associated with atherosclerosis, fibrotic disorders,pulmonary fibrosis, arthritis, rheumatoid arthritis, glomerulonephritis,and post-surgical stenosis, restenosis, disorders of proliferation ofblood vessels, disorders of proliferation of mesangial cells, metabolicdisorders, allergies, asthmas, thromboses, diseases of the nervoussystem, retinopathy, diabetes, and muscular degeneration. In certainembodiments, the proliferative disorder is cancer.

In certain embodiments, the cancer is selected from the group consistingof cancer of the bone, brain, connective tissue, endocrine glands,adrenal cortex, endometrium, germ cells, head and neck, larynx andhypopharynx, mesothelioma, muscle, rectum, renal, small intestine, softtissue, testis, ureter, vagina, and vulva; bladder cancer; breastcancer; colon cancer; kidney cancer; liver cancer; lung cancer;esophagus cancer; gallbladder cancer; ovarian cancer; pancreatic cancer;stomach cancer; cervical cancer; thyroid cancer; prostate cancer;papillary thyroid carcinoma; genitourinary malignancies; retinoblastoma;Wilms tumor; myelodysplastic syndrome; plasma cell neoplasia;paraneoplastic syndromes; renal cell carcinoma; Ewing's sarcoma;desmoplastic small round cell tumors; mesothelioma; skin cancer, whereinsaid skin cancer is squamous cell carcinoma; hematologic cancers [e.g.,hematopoietic cancers of lymphoid lineage, wherein said cancers areleukemia, acute lymphocytic leukemia (ALL), acute lymphoblasticleukemia, lymphoma, B-cell lymphoma, T-cell lymphoma, Hodgkin'slymphoma, non-Hodgkin's lymphoma (e.g., mantle cell lymphoma (MCL),hairy cell lymphoma, and Burkitt's lymphoma; chronic lymphocyticleukemia (CLL); hematopoietic cancers of myeloid lineage, wherein saidcancers are multiple myeloma, chronic myeloid leukemia (CML) and acutemyeloid leukemia (AML) (e.g., acute megakaryoblastic leukemia (AMKL);myelodysplastic syndrome and promyelocytic leukemia]; tumors ofmesenchymal origin, wherein the tumors are fibrosarcoma andrhabdomyosarcoma; tumors of the central and peripheral nervous system,wherein said tumors are astrocytoma, neuroblastoma, glioma (e.g.,glioblastoma) and schwannomas; and other tumors, wherein said tumors aremelanoma, cutaneous melanoma, seminoma, teratocarcinoma, osteosarcoma,xeroderma pegmentosum, keratoxanthoma, thyroid follicular cancer,Kaposi's sarcoma, cancers of unknown primary site; solid tumors,hematologic cancers, and AIDS-related malignancies.

In certain embodiments, the cancer is a multi-drug resistant (MDR)cancer.

In certain embodiments, the cancer is relapsed and/or refractory cancer.Relapsed cancer refers to a cancer which has returned after a patienthas enjoyed a remission. Refractory cancer refers to a cancer which doesnot respond to other therapies or therapeutic agents. In certainembodiments, the cancer is resistant to (i.e., does not respond to)therapies or chemotherapeutic agents. In certain embodiments, thehematologic cancer is resistant to therapies or chemotherapeutic agents.

In certain embodiments, the cancer is leukemia, lymphoma, melanoma,cancer of the breast, stomach, ovaries, colon, rectum, lung, brain,larynx, lymphatic system, thyroid, oesophagus, liver, uterus, testis,bladder, prostate, bones or pancreas. In certain embodiments, the canceris leukemia, cancer of the breast, the colon or the lung.

In certain embodiments, the cancer is a hematologic cancer. In certainembodiments, the compound inhibits the growth of hematologic cancers. Incertain embodiments, the compound inhibits the growth of hematologiccancers with IC50s in the nanomolar range. In certain embodiments, thehematologic cancer is a hematopoietic cancer of lymphoid lineage. Incertain embodiments, the hematologic cancer is a relapsed and/orrefractory hematopoietic cancer of lymphoid lineage. In certainembodiments, the cancer is refractory to multiple cycles of cancertherapy (e.g., including allogenic bone marrow transplantation). Incertain embodiments, the hematologic cancer is relapsed and/orrefractory ALL, CLL, AML, or CML.

In certain embodiments, the cancer is ovarian cancer. In certainembodiments, the ovarian cancer is ovarian carcinoma.

In certain embodiments, the cancer is lung cancer. In certainembodiments, the lung cancer is non-small cell lung cancer. In certainembodiments, the lung cancer is small cell lung cancer. In certainembodiments, the lung cancer is lung adenocarcinoma.

In certain embodiments, the cancer is prostate cancer.

In certain embodiments, the cancer is renal cell carcinoma.

In certain embodiments, the cancer is cervical cancer. In certainembodiments, the cervical cancer is cervical adenocarcinoma. In certainembodiments, the cervical cancer is positive for human papillomavirus(HPV) infection.

In certain embodiments, the cancer is glioblastoma.

In certain embodiments, the cancer is retinoblastoma.

In certain embodiments, the cancer is rhabdomyosarcoma.

In certain embodiments, the cancer is a desmoplastic small round celltumor.

In certain embodiments, the cancer is breast cancer. In certainembodiments, the breast cancer is breast ductal carcinoma. In certainembodiments, the breast cancer is breast adenocarcinoma. In certainembodiments, the breast cancer is metastatic breast adenocarcinoma. Incertain embodiments, the breast cancer is HER2 negative. In certainembodiments, the breast cancer is HER2 positive. In certain embodiments,the breast cancer is NEU receptor negative.

In certain embodiments, the cancer is mesothelioma.

In certain embodiments, the cancer is melanoma.

In certain embodiments, the cancer is thyroid carcinoma.

In certain embodiments, the cancer is Ewing's sarcoma.

In certain embodiments, the cancer is a solid tumor. In certainembodiments, the compound inhibits the growth of solid tumors. Incertain embodiments, the compound inhibits the growth of solid tumorswith IC50s in the nanomolar range.

In certain embodiments, the cancer comprises a genetic mutation.

In certain embodiments, the cancer comprises a RAS mutation. In certainembodiments, the cancer comprises wild-type RAS.

In certain embodiments, cancer comprises an EGFR mutation. In certainembodiments, the EGFR mutation is an L858R EGFR mutation. In certainembodiments, the EGFR mutation is an DelE746 EGFR mutation. In certainembodiments, the EGFR mutation is an DelE746-A750 EGFR mutation. Incertain embodiments, the EGFR mutation is an DelE746-E749 EGFR mutation.In certain embodiments, the EGFR mutation is an T790M EGFR mutation. Incertain embodiments, the EGFR mutation is an T790M/L858R EGFR mutation.In certain embodiments, the cancer comprises wild-type EGFR.

In certain embodiments, the cancer comprises a KRAS mutation. In certainembodiments, the cancer comprises a G13C KRAS mutation. In certainembodiments, the cancer comprises a G12C KRAS mutation. In certainembodiments, the cancer comprises a G12C KRAS mutation. In certainembodiments, the cancer comprises a Q61H KRAS mutation. In certainembodiments, the cancer comprises wild-type KRAS.

In certain embodiments, the cancer comprises a p53 mutation. In certainembodiments, the p53 mutation is a R273H p53 mutation. In certainembodiments, the p53 mutation is a G262V p53 mutation. In certainembodiments, the p53 mutation is a G16L p53 mutation. In certainembodiments, the p53 mutation is a C176F p53 mutation. In certainembodiments, the p53 mutation is a M246I p53 mutation. In certainembodiments, the cancer comprises wild-type p53.

In certain embodiments, the cancer comprises a BRAF mutation. In certainembodiments, the BRAF mutation is a BRAF V600E mutation.

In certain embodiments, the cancer comprises a EVI1 mutation.

In certain embodiments, the cancer comprises a Flt-3 mutation.

In certain embodiments, the cancer comprises a WT-1 mutation.

In certain embodiments, the cancer comprises a cyclin D mutation.

In certain embodiments, the cancer comprises a PTEN mutation.

In certain embodiments, the cancer comprises a ABL kinase mutation.

In certain embodiments, the mutation comprises a chromosomalabnormality. In certain embodiments, the chromosomal abnormality is achromosome deletion or inversion. In certaine embodiments, the cancercomprises a chromosome 17p deletion. In certain embodiments, the cancercomprises an inversion of chromosome 16. In certain embodiments, thecancer comprises a trisomy of chromosome 8. In certain embodiments, thecancer comprises a monosomy of chromosome 7. In certain embodiments, thecancer comprises a chromosome 11q23 abnormality. In certain embodiments,the cancer comprises a Philadelphia chromosome positive abnormality.

In certain embodiments, the cancer comprises a fusion transcript. Incertain embodiments, the fusion transcript is a reciprocal ASPL-TFE3fusion transcript.

Assays

In some embodiments, the present invention provides a method ofdetermining dose response using a cytotoxicity assay. In someembodiments, the present invention includes a method of performing doseresponse studies comprising the steps of providing a test compound,contacting the test compound with a cell, and incubating the cell withthe compound under suitable conditions to determine the cytotoxicity ofthe compound. The antiproliferative activity of the test compound canthen be assessed using a method known to those of ordinary skill in theart. This process can then be repeated using different concentrations ofa test compound in order to calculate the IC₅₀. In certain embodiments,the test compound is an compound. In certain embodiments, the cells areretinoblastoma cells.

In certain embodiments, the cells are incubated with a test compound(e.g., an compound) for approximately 1 minute to approximately 1 week.In certain embodiments, the cells are incubated with a test compound forapproximately 1 hour to approximately 1 week. In certain embodiments,the cells are incubated with a test compound for approximately 12 hoursto approximately 1 week. In certain embodiments, the cells are incubatedwith a test compound for approximately 24 hours to approximately 1 week.In certain embodiments, the cells are incubated with a test compound forapproximately 36 hours to approximately 1 week. In certain embodiments,the cells are incubated with a test compound for approximately 48 hoursto approximately 1 week. In certain embodiments, the cells are incubatedwith a test compound for approximately 48 hours to approximately 120hours. In certain embodiments, the cells are incubated with a testcompound for approximately 48 hours to approximately 96 hours. Incertain embodiments, the cells are incubated with a test compound forapproximately 62 hours to approximately 82 hours. In certainembodiments, the cells are incubated with a test compound forapproximately 72 hours. In certain embodiments, the cells are incubatedwith a test compound for approximately 1, 2, 3, 4, 5, 6, or 7 days.

In certain embodiments, after a specified amount of time (e.g., 72hours) an indicator of cell viability (e.g., Alamar Blue) is added, andthe mixture is incubated for an additional period of time. In someembodiments, this additional period of time ranges from approximately 1hour to approximately 48 hours. In some embodiments, this additionalperiod of time ranges from approximately 12 hour to approximately 36hours. In some embodiments, this additional period of time isapproximately 24 hours.

The inhibition of cell proliferation may be measured using methods ortechnology known in the art. In some embodiments, inhibition of cellproliferation is measured using a substance which produces a detectablesignal that is proportional to the amount of inhibition of cellproliferation. In some embodiments, inhibition of cell proliferation isquantified using one of any indicators known to those of ordinary skillin the art that produces a quantifiable signal, the intensity of whichis detectable and proportional to the amount of inhibition. In someembodiments, inhibition of cell proliferation is quantified using anindicator which fluoresces. Exemplary indicators include Tyramide-AlexaFluor 488, Alamar Blue, etc.

In some embodiments, the efficacy of a compound is measured by measuringtumor size over a period of time before, during, and/or after treatmentwith said compound. In some embodiments, tumor size is measured once aweek. In some embodiments, tumor size is measured twice a week. In someembodiments, tumor size is measured daily. In some embodiments, tumorsize is measured once a day. In some embodiments, tumor size is measuredtwice a day. In some embodiments, tumor size is measured once everyother day. In some embodiments, tumor size is measured once every threedays. In certain embodiments, tumor size is measured at intervals asrequired by any one of the methods known to those of skill in the art.In some embodiments, tumor size is measured externally twice a week witha caliper. In certain embodiments, tumor size is measured once a weekusing an imaging technique (e.g., MRI, X-ray, CT). In some embodiments,the imaging technique is bioluminescent imaging. In certain embodiments,bioluminescent imaging comprises anesthetization of the host animal,injection of a bioluminescent compound, and subsequent measurement ofphotonic emission. In some embodiments, imaging of the tumor is achievedusing any of the methods known in the medical arts.

As detailed herein, in assays to determine the ability of a compound(e.g., an compound) to inhibit cancer cell growth certain compounds mayexhibit IC₅₀ values ≦100 μM. In certain other embodiments, the compoundexhibits IC₅₀ values ≦50 μM. In certain other embodiments, the compoundexhibits IC₅₀ values ≦40 μM. In certain other embodiments, the compoundexhibits IC₅₀ values ≦30 μM. In certain other embodiments, the compoundexhibits IC₅₀ values ≦20 μM. In certain other embodiments, the compoundexhibits IC₅₀ values ≦10 μM. In certain other embodiments, the compoundexhibits IC₅₀ values ≦7.5 μM. In certain embodiments, the compoundexhibits IC₅₀ values ≦5 μM. In certain other embodiments, the compoundexhibits IC₅₀ values ≦2.5 μM. In certain embodiments, the compoundexhibits IC₅₀ values ≦1 μM. In certain embodiments, the compoundexhibits IC₅₀ values ≦0.75 μM. In certain embodiments, the compoundexhibits IC₅₀ values ≦0.5 μM. In certain embodiments, the compoundexhibits IC₅₀ values ≦0.25 μM. In certain embodiments, the compoundexhibits IC₅₀ values ≦0.1 μM. In certain other embodiments, the compoundexhibits IC₅₀ values ≦75 nM. In certain other embodiments, the compoundexhibits IC₅₀ values ≦50 nM. In certain other embodiments, the compoundexhibits IC₅₀ values ≦25 nM. In certain other embodiments, the compoundexhibits IC₅₀ values ≦10 nM. In other embodiments, the compound exhibitsIC₅₀ values ≦7.5 nM. In other embodiments, the compound exhibits IC₅₀values ≦5 nM.

Pharmaceutical Compositions

Certain aspects of the invention include use of a pharmaceuticalcomposition in any of the above methods, comprising a compound ofFormula (A) or (B) or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable excipient.

Pharmaceutically acceptable excipients include any and all solvents,diluents or other liquid vehicles, dispersion or suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, solid binders, lubricants and the like, as suitedto the particular dosage form desired. General considerations in theformulation and/or manufacture of pharmaceutical compositions agents canbe found, for example, in Remington's Pharmaceutical Sciences, SixteenthEdition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), andRemington: The Science and Practice of Pharmacy, 21^(st) Edition(Lippincott Williams & Wilkins, 2005).

Pharmaceutical compositions described herein can be prepared by anymethod known in the art of pharmacology. In general, such preparatorymethods include the steps of bringing the active ingredient intoassociation with a carrier and/or one or more other accessoryingredients, and then, if necessary and/or desirable, shaping and/orpackaging the product into a desired single- or multi-dose unit.

Pharmaceutical compositions can be prepared, packaged, and/or sold inbulk, as a single unit dose, and/or as a plurality of single unit doses.As used herein, a “unit dose” is discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient which would be administered to a subject and/or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and/or any additional ingredients in apharmaceutical composition of the invention will vary, depending uponthe identity, size, and/or condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.By way of example, the composition may comprise between 0.1% and 100%(w/w) active ingredient.

Pharmaceutically acceptable excipients used in the manufacture ofprovided pharmaceutical compositions include inert diluents, dispersingand/or granulating agents, surface active agents and/or emulsifiers,disintegrating agents, binding agents, preservatives, buffering agents,lubricating agents, and/or oils. Excipients such as cocoa butter andsuppository waxes, coloring agents, coating agents, sweetening,flavoring, and perfuming agents may also be present in the composition.

Exemplary diluents include calcium carbonate, sodium carbonate, calciumphosphate, dicalcium phosphate, calcium sulfate, calcium hydrogenphosphate, sodium phosphate lactose, sucrose, cellulose,microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodiumchloride, dry starch, cornstarch, powdered sugar, etc., and combinationsthereof.

Exemplary granulating and/or dispersing agents include potato starch,corn starch, tapioca starch, sodium starch glycolate, clays, alginicacid, guar gum, citrus pulp, agar, bentonite, cellulose and woodproducts, natural sponge, cation-exchange resins, calcium carbonate,silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone)(crospovidone), sodium carboxymethyl starch (sodium starch glycolate),carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose(croscarmellose), methylcellulose, pregelatinized starch (starch 1500),microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate,quaternary ammonium compounds, etc., and combinations thereof.

Exemplary surface active agents and/or emulsifiers include naturalemulsifiers (e.g. acacia, agar, alginic acid, sodium alginate,tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk,casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g.bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]),long chain amino acid derivatives, high molecular weight alcohols (e.g.stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate,ethylene glycol distearate, glyceryl monostearate, and propylene glycolmonostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene,polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer),carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium,powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acidesters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20],polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate[Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate[Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitanmonooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylenemonostearate [Myrj 45], polyoxyethylene hydrogenated castor oil,polyethoxylated castor oil, polyoxymethylene stearate, and Solutol),sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g.Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether[Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate,triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate,oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68,Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride,benzalkonium chloride, docusate sodium, etc. and/or combinationsthereof.

Exemplary binding agents include starch (e.g. cornstarch and starchpaste), gelatin, sugars (e.g. sucrose, glucose, dextrose, dextrin,molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums(e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghattigum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose,ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, microcrystalline cellulose, celluloseacetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum),and larch arabogalactan), alginates, polyethylene oxide, polyethyleneglycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes,water, alcohol, etc., and/or combinations thereof.

Exemplary preservatives include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene,monothioglycerol, potassium metabisulfite, propionic acid, propylgallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, andsodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid(EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodiumedetate, trisodium edetate, calcium disodium edetate, dipotassiumedetate, and the like), citric acid and salts and hydrates thereof(e.g., citric acid monohydrate), fumaric acid and salts and hydratesthereof, malic acid and salts and hydrates thereof, phosphoric acid andsalts and hydrates thereof, and tartaric acid and salts and hydratesthereof. Exemplary antimicrobial preservatives include benzalkoniumchloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide,cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol,chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea,phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate,propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methylparaben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoicacid, potassium benzoate, potassium sorbate, sodium benzoate, sodiumpropionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol,phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate,and phenylethyl alcohol.

Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E,beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbicacid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroximemesylate, cetrimide, butylated hydroxyanisol (BHA), butylatedhydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus,Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, andEuxyl. In certain embodiments, the preservative is an anti-oxidant. Inother embodiments, the preservative is a chelating agent.

Exemplary buffering agents include citrate buffer solutions, acetatebuffer solutions, phosphate buffer solutions, ammonium chloride, calciumcarbonate, calcium chloride, calcium citrate, calcium glubionate,calcium gluceptate, calcium gluconate, D-gluconic acid, calciumglycerophosphate, calcium lactate, propanoic acid, calcium levulinate,pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasiccalcium phosphate, calcium hydroxide phosphate, potassium acetate,potassium chloride, potassium gluconate, potassium mixtures, dibasicpotassium phosphate, monobasic potassium phosphate, potassium phosphatemixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodiumcitrate, sodium lactate, dibasic sodium phosphate, monobasic sodiumphosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide,aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline,Ringer's solution, ethyl alcohol, etc., and combinations thereof.

Exemplary lubricating agents include magnesium stearate, calciumstearate, stearic acid, silica, talc, malt, glyceryl behanate,hydrogenated vegetable oils, polyethylene glycol, sodium benzoate,sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate,sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include almond, apricot kernel, avocado, babassu,bergamot, black current seed, borage, cade, camomile, canola, caraway,carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee,corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed,geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate,jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademianut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, andwheat germ oils. Exemplary oils include, but are not limited to, butylstearate, caprylic triglyceride, capric triglyceride, cyclomethicone,diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil,octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.

Liquid dosage forms for oral and parenteral administration includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active ingredients,the liquid dosage forms may comprise inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can include adjuvants such as wetting agents, emulsifyingand suspending agents, sweetening, flavoring, and perfuming agents. Incertain embodiments for parenteral administration, the conjugates of theinvention are mixed with solubilizing agents such as Cremophor,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can be a sterile injectable solution,suspension or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This can be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing the conjugates of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may comprise buffering agents.

Solid compositions of a similar type can be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type can be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active ingredients can be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active ingredient can be admixed with at least oneinert diluent such as sucrose, lactose or starch. Such dosage forms maycomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may comprise bufferingagents. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of a compoundof this invention may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants and/or patches. Generally, theactive ingredient is admixed under sterile conditions with apharmaceutically acceptable carrier and/or any needed preservativesand/or buffers as can be required. Additionally, the present inventioncontemplates the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of an active ingredient tothe body. Such dosage forms can be prepared, for example, by dissolvingand/or dispensing the active ingredient in the proper medium.Alternatively or additionally, the rate can be controlled by eitherproviding a rate controlling membrane and/or by dispersing the activeingredient in a polymer matrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositionscan be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 and functional equivalents thereof. Jetinjection devices which deliver liquid vaccines to the dermis via aliquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable. Jetinjection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable. Alternatively or additionally, conventionalsyringes can be used in the classical mantoux method of intradermaladministration.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient can be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention can be prepared, packaged,and/or sold in a formulation suitable for pulmonary administration viathe buccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers or from about 1 to about 6nanometers. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant can be directed to dispersethe powder and/or using a self propelling solvent/powder dispensingcontainer such as a device comprising the active ingredient dissolvedand/or suspended in a low-boiling propellant in a sealed container. Suchpowders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositions mayinclude a solid fine powder diluent such as sugar and are convenientlyprovided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic and/or solid anionic surfactant and/or a solid diluent(which may have a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may provide the active ingredient in the form of droplets of asolution and/or suspension. Such formulations can be prepared, packaged,and/or sold as aqueous and/or dilute alcoholic solutions and/orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization and/oratomization device. Such formulations may further comprise one or moreadditional ingredients including, but not limited to, a flavoring agentsuch as saccharin sodium, a volatile oil, a buffering agent, a surfaceactive agent, and/or a preservative such as methylhydroxybenzoate. Thedroplets provided by this route of administration may have an averagediameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare useful for intranasal delivery of a pharmaceutical composition ofthe invention. Another formulation suitable for intranasaladministration is a coarse powder comprising the active ingredient andhaving an average particle from about 0.2 to 500 micrometers. Such aformulation is administered. by rapid inhalation through the nasalpassage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition of theinvention can be prepared, packaged, and/or sold in a formulationsuitable for buccal administration. Such formulations may, for example,be in the form of tablets and/or lozenges made using conventionalmethods, and may contain, for example, 0.1 to 20% (w/w) activeingredient, the balance comprising an orally dissolvable and/ordegradable composition and, optionally, one or more of the additionalingredients described herein. Alternately, formulations suitable forbuccal administration may comprise a powder and/or an aerosolized and/oratomized solution and/or suspension comprising the active ingredient.Such powdered, aerosolized, and/or aerosolized formulations, whendispersed, may have an average particle and/or droplet size in the rangefrom about 0.1 to about 200 nanometers, and may further comprise one ormore of the additional ingredients described herein.

A pharmaceutical composition of the invention can be prepared, packaged,and/or sold in a formulation suitable for ophthalmic administration.Such formulations may, for example, be in the form of eye dropsincluding, for example, a 0.1/1.0% (w/w) solution and/or suspension ofthe active ingredient in an aqueous or oily liquid carrier. Such dropsmay further comprise buffering agents, salts, and/or one or more otherof the additional ingredients described herein. Otheropthalmically-administrable formulations which are useful include thosewhich comprise the active ingredient in microcrystalline form and/or ina liposomal preparation. Ear drops and/or eye drops are contemplated asbeing within the scope of this invention.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with ordinary experimentation.General considerations in the formulation and/or manufacture ofpharmaceutical compositions can be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005.

Provided compounds can be administered using any amount and any route ofadministration effective for treatment. The exact amount required willvary from subject to subject, depending on the species, age, and generalcondition of the subject, the severity of the infection, the particularcomposition, its mode of administration, its mode of activity, and thelike.

Compounds provided herein are typically formulated in dosage unit formfor ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention will be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective dose level for any particular subject or organism will dependupon a variety of factors including the disease, disorder, or conditionbeing treated and the severity of the disorder; the activity of thespecific active ingredient employed; the specific composition employed;the age, body weight, general health, sex and diet of the subject; thetime of administration, route of administration, and rate of excretionof the specific active ingredient employed; the duration of thetreatment; drugs used in combination or coincidental with the specificactive ingredient employed; and like factors well known in the medicalarts.

The compounds and compositions provided herein can be administered byany route, including oral, intravenous, intramuscular, intra-arterial,intramedullary, intrathecal, subcutaneous, intraventricular,transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical(as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal,enteral, sublingual; by intratracheal instillation, bronchialinstillation, and/or inhalation; and/or as an oral spray, nasal spray,and/or aerosol. Specifically contemplated routes are systemicintravenous injection, regional administration via blood and/or lymphsupply, and/or direct administration to an affected site, in a bolus orcontinuous infusion regimen. In general the most appropriate route ofadministration will depend upon a variety of factors including thenature of the agent (e.g., its stability in the environment of thegastrointestinal tract), the condition of the subject (e.g., whether thesubject is able to tolerate oral administration), etc.

The exact amount of a compound required to achieve a therapeuticallyeffective amount will vary from subject to subject, depending, forexample, on species, age, and general condition of a subject, severityof the side effects or disorder, identity of the particular compound(s),mode of administration, and the like. The desired dosage can bedelivered three times a day, two times a day, once a day, every otherday, every third day, every week, every two weeks, every three weeks, orevery four weeks. In certain embodiments, the desired dosage can bedelivered using multiple administrations (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, ormore administrations).

In certain embodiments, a therapeutically effective amount of a compoundfor administration one or more times a day to a 70 kg adult human maycomprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg,about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about1000 mg, or about 100 mg to about 1000 mg, about 0.001 mg to about 100mg, from about 0.01 mg to about 50 mg, from about 0.1 mg to about 40 mg,from about 0.5 mg to about 30 mg, from about 0.01 mg to about 10 mg,from about 0.1 mg to about 10 mg, and from about 1 mg to about 25 mg,per kilogram, of a compound. It will be appreciated that dose ranges asdescribed herein provide guidance for the administration of providedpharmaceutical compositions to an adult. The amount to be administeredto, for example, a child or an adolescent can be determined by a medicalpractitioner or person skilled in the art and can be lower or the sameas that administered to an adult. The desired dosage may be deliveredthree times a day, two times a day, once a day, every other day, everythird day, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage may be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations).

The method comprises administration of a compound in a therapeuticallyeffective dose to a host subject. In some embodiments, the subject is ananimal. In some embodiments, a therapeutically effective dose comprisesan amount ranging from approximately 0.1 mg/kg to approximately 50.0mg/kg. In some embodiments, a therapeutically effective dose comprisesan amount ranging from approximately 0.5 mg/kg to approximately 50.0mg/kg. In some embodiments, a therapeutically effective dose comprisesan amount ranging from approximately 0.5 mg/kg to approximately 40.0mg/kg. In some embodiments, a therapeutically effective dose comprisesan amount ranging from approximately 0.5 mg/kg to approximately 30.0mg/kg. In some embodiments, a therapeutically effective dose comprisesan amount ranging from approximately 1.0 mg/kg to approximately 25.0mg/kg. In some embodiments, a therapeutically effective dose comprisesan amount ranging from approximately 1.5 mg/kg to approximately 15.0mg/kg. In some embodiments, treatment is administered locally. In someembodiments, treatment is administered by continuous infusion over acertain period of time. In certain embodiments, administration is viaintraarterial infusion. In certain embodiments, administration is viaintraarterial infusion via an artery feeding the tumor being treated.

Some embodiments of the invention further comprise the administration ofat least one other therapy or therapeutic agents. The compound orcomposition can be administered concurrently with, prior to, orsubsequent to, the other therapy or therapeutic agents. In general, eachtherapy or agent will be administered at a dose and/or on a timeschedule determined for that agent. In will further be appreciated thatthe additional therapeutically active agent utilized in this combinationcan be administered together in a single composition or administeredseparately in different compositions. The particular combination toemploy in a regimen will take into account compatibility of the compoundwith the therapy or therapeutic agent and/or the desired therapeuticeffect to be achieved. In general, it is expected that additionaltherapy or therapeutic agent utilized in combination be utilized atlevels that do not exceed the levels at which they are utilizedindividually. In some embodiments, the levels utilized in combinationwill be lower than those utilized individually.

The compounds or compositions can be administered in combination with atherapy or therapeutic agent that improves their bioavailability, reduceand/or modify their metabolism, inhibit their excretion, and/or modifytheir distribution within the body. It will also be appreciated that thetherapy or therapeutic agent employed may achieve a desired effect forthe same disease or disorder, and/or it may achieve different effects(e.g., control of adverse side-effects).

Cancer therapies include, but are not limited to, surgery and surgicaltreatments, radiation therapy, and therapeutic agents (e.g.,biotherapeutic agents and chemotherapeutic agents). In certainembodiments, the method comprises administration of radiation.

Exemplary biotherapeutic agents include, but are not limited to,interferons, cytokines (e.g., tumor necrosis factor, interferon α,interferon γ), vaccines, hematopoietic growth factors, monoclonalserotherapy, immunostimulants and/or immunodulatory agents (e.g., IL-1,2, 4, 6, or 12), immune cell growth factors (e.g., GM-CSF) andantibodies (e.g. HERCEPTIN (trastuzumab), T-DM1, AVASTIN (bevacizumab),ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN (rituximab), BEXXAR(tositumomab)).

Exemplary chemotherapeutic agents include, but are not limited to,anti-estrogens (e.g. tamoxifen, raloxifene, and megestrol), LHRHagonists (e.g. goscrclin and leuprolide), anti-androgens (e.g. flutamideand bicalutamide), photodynamic therapies (e.g. vertoporfin (BPD-MA),phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A(2BA-2-DMHA)), nitrogen mustards (e.g. cyclophosphamide, ifosfamide,trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas(e.g. carmustine (BCNU) and lomustine (CCNU)), alkylsulphonates (e.g.busulfan and treosulfan), triazenes (e.g. dacarbazine, temozolomide),platinum containing compounds (e.g. cisplatin, carboplatin,oxaliplatin), vinca alkaloids (e.g. vincristine, vinblastine, vindesine,and vinorelbine), taxoids (e.g. paclitaxel or a paclitaxel equivalentsuch as nanoparticle albumin-bound paclitaxel (ABRAXANE),docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin),polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex,CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2bound to three molecules of paclitaxel), paclitaxel-EC-1 (paclitaxelbound to the erbB2-recognizing peptide EC-1), and glucose-conjugatedpaclitaxel, e.g., 2′-paclitaxel methyl 2-glucopyranosyl succinate;docetaxel, taxol), epipodophyllins (e.g. etoposide, etoposide phosphate,teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan,irinotecan, crisnatol, mytomycin C), anti-metabolites, DHFR inhibitors(e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMPdehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin,and EICAR), ribonuclotide reductase inhibitors (e.g. hydroxyurea anddeferoxamine), uracil analogs (e.g. 5-fluorouracil (5-FU), floxuridine,doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosineanalogs (e.g. cytarabine (ara C), cytosine arabinoside, andfludarabine), purine analogs (e.g. mercaptopurine and Thioguanine),Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylationinhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g.1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g.staurosporine), actinomycin (e.g. actinomycin D, dactinomycin),bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline(e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin,idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDRinhibitors (e.g. verapamil), Ca²⁺ ATPase inhibitors (e.g. thapsigargin),imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g.,axitinib (AG13736), bosutinib (SKI-606), cediranib (RECENTIN™, AZD2171),dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib(IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®,TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib(TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®, SU11248),toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474), vatalanib (PTK787,PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab(RITUXAN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), ranibizumab(Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), everolimus(AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumab ozogamicin (MYLOTARG®),temsirolimus (TORISEL®), ENMD-2076, PCI-32765, AC220, dovitinib lactate(TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903,PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120(VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154,CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and/orXL228), proteasome inhibitors (e.g., bortezomib (VELCADE)), mTORinhibitors (e.g., rapamycin, temsirolimus (CCI-779), everolimus(RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235(Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502(Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)),oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed,cyclophosphamide, dacarbazine, procarbizine, prednisolone,dexamethasone, campathecin, plicamycin, asparaginase, aminopterin,methopterin, porfiromycin, melphalan, leurosidine, leurosine,chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin,aminopterin, and hexamethyl melamine.

Kits

In still another aspect, the present invention also provides kits (e.g.,pharmaceutical packs) for treating a proliferative disorder comprising acompound of the Formula (A) or (B) and instructions for administrationto a subject for treating a proliferative disorder. Kits may comprise aprovided composition and a container (e.g., a vial, ampoule, bottle,syringe, and/or dispenser package, or other suitable container). In someembodiments, provided kits may optionally further include a secondcontainer comprising a suitable aqueous carrier for dilution orsuspension of the provided composition for preparation of administrationto a subject. In some embodiments, contents of provided formulationcontainer and solvent container combine to form at least one unit dosageform.

Optionally, a single container may comprise one or more compartments forcontaining a provided composition, and/or appropriate aqueous carrierfor suspension or dilution. In some embodiments, a single container canbe appropriate for modification such that the container may receive aphysical modification so as to allow combination of compartments and/orcomponents of individual compartments. For example, a foil or plasticbag may comprise two or more compartments separated by a perforated sealwhich can be broken so as to allow combination of contents of twoindividual compartments once the signal to break the seal is generated.A pharmaceutical pack or kit may thus comprise such multi-compartmentcontainers including a provided composition and appropriate solventand/or appropriate aqueous carrier for suspension.

Optionally, instructions for use are additionally provided in such kitsof the invention. Such instructions may provide, generally, for example,instructions for dosage and administration. In other embodiments,instructions may further provide additional detail relating tospecialized instructions for particular containers and/or systems foradministration. Still further, instructions may provide specializedinstructions for use in conjunction and/or in combination withadditional therapy.

EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

Example 1. Inhibition of Primary Patient Samples

Table 1 highlights a series of primary patient leukemia samples-bothacute and chronic and treatment naïve and refractory and the effect ofgranaticin A, granaticin B, derivative 1, and derivative 2 to inhibitcellular proliferation. The IC₅₀ data reveals that granaticin B isapproximately one log more active than granaticin A against all thesamples tested and inhibits cellular proliferation in the low nanomolarrange. The two starred samples represent serial samples taken from thesame patients after they received 2-3 cycles of additional high dosesalvage chemotherapy and indicate that although refractory to salvagechemotherapy, the Cdc7 pathway remains a potentially efficacious targetas the sensitivity to these compounds remains unchanged. Importantly,each patient sample was resistant to the prior therapy received usingthis assay.

TABLE 1 Inhibition of patient leukemia samples IC₅₀ (microM) GranaticinDeriv. Deriv. Granaticin Leukemia sample A 1 2 B AML, de novo 0.72 0.622.01 0.18 AML, primary refractory 1 0.75 0.62 1.55 0.11 AML, primaryrefractory 1* 0.63 0.47 1.44 0.10 AML, primary refractory 0.77 0.84 1.450.14 AML, relapsed 1.20 0.70 2.23 0.10 AML, relapsed 0.21 0.23 0.35 0.05AML, relapsed s/p HSCT 1.88 1.86 8.10 0.62 AMMoL 0.99 0.79 5.88 0.49ALL, Ph+ 0.56 0.47 2.57 0.19 Biphenotypic acute 0.30 0.69 0.90 0.04leukemia 1 Biphenotypic acute 0.54 0.76 1.02 0.10 leukemia 1* CML,untreated 0.26 0.33 1.24 0.08 CLL, untreated, 0.46 0.36 1.21 0.31chromosome 17p del. PhALL3.1 0.14 0.12 0.47 0.03 *Indicates a secondsample from the same patient following high-dose chemotherapy

Table 2 highlights cell lines and primary patient samples tested forgranaticin A and granaticin B to inhibit cellular proliferation(summarized from FIGS. 16A and 16B).

TABLE 2 IC₅₀ (microM) Cell Line Description Granaticin A Granaticin BALL3 Hematopoietic-Human 0.14 0.03 Acute Lymphoblastic leukemia;Philadelphia chromosome positive HL60 Hematopoietic-Human 0.14 0.09Acute Promyelocytic leukemia HL60 MX1 Hematopoietic-Human 0.17 0.08Acute Promyelocytic leukemia; MDR variant of HL60 selected withmitoxantrone-clone 1 HL60 MX2 Hematopoietic-Human 0.11 0.07 AcutePromyelocytic leukemia; MDR variant of HL60 selected withmitoxantrone-clone 2 HL60 RV Hematopoietic-Human 0.09 0.07 AcutePromyelocytic leukemia: MDR variant of HL60 selected with vincristineJEKO Hematopoietic-Human 0.04 0.02 Mantle Cell Lymphoma JURKAT E61Hematopoietic-Human 0.05 0.02 Acute Lymphoblastic T- cell leukemia K562Hematopoietic-Human 0.12 0.08 Chronic Myelogenous leukemia transformedto acute erythroleukemia KASUMI4 Hematopoietic-Human 0.30 0.18 ChronicMyelogenous leukemia transformed to acute myeloid leukemia-overexpression of EVI1 MEG01 Hematopoietic-Human 0.17 0.08 ChronicMyelogenous leukemia transformed to acute Megakaryoblastic leukemiaMOLT3 Hematopoietic-Human 0.03 0.01 Acute Lympholastic T-cell LeukemiaNCEB1 Hematopoietic-Human 0.33 0.26 Mantle Cell Lymphoma 132Primary-Patient sample of 0.13 0.01 Chronic Lymphocytic Leukemia ALBUPrimary-Patient sample of 0.43 0.03 essential thrombocythemiatransformed to refractory acute myelogenous leukemia DOGOPrimary-Patient sample of 0.15 0.07 Chronic Lymphocytic Leukemia withtrisomy of chromosome 12 and unmutated Ig heavy chain locus GLHUPrimary-Patient sample of 0.26 0.08 Chronic Myelogenous leukemia JABRPrimary-Patient sample of 0.21 0.05 relpased/refractory acutemyelogenous leukemia with normal cytogenetics and Flt-3 ITD JAKLPrimary-Patient sample of 0.05 0.01 Chronic Lymphocytic Leukemia withtrisomy of chromosome 12 and unmutated Ig heavy chain locus JAMCPrimary-Patient sample of 0.06 0.04 acute myelogenous leukemia withinversion of chromosome 16 JAQU Primary-Patient sample of 0.05 0.01relapsed/refractory pre-B cell acute lymphoblastic leukemia JOBLPrimary-Patient sample of 0.56 0.19 relapsed/refractory acutemyelogenous leukemia JOHO Primary-Patient sample of 0.88 0.17 primaryrefractory acute myelogenous leukemia with inv3 and monosomy 7(refractory to allogeneic BMT) JOQU Primary-Patient sample of 0.77 0.14relapsed/refractory acute myelogenous leukemia with trisomy ofchromosome #8 and Flt-3 ITD JUCO Primary-Patient sample of 0.08 0.01pre-B cell acute lymphoblastic leukemia with t(4;11) involving the MLLgene MAWI Primary-Patient sample of 0.72 0.18 relpased/refractory acutemyelogenous leukemia with normal cytogenetics MAWI B Primary-Patientsample of 0.42 0.16 secondary chronic myelogenous leukemia MIHAPrimary-Patient sample of 0.30 0.04 relapsed/refractory acutebiphenotypic leukemia (patient was refractory to allogeneic bone marrowtransplant) PAPR Primary-Patient sample of 0.99 0.49 relapsed acutemyelomonocytic leukemia RADO Primary-Patient sample of 0.06 0.03 primaryrefractory acute myelogenous leukemia with multiple chromosomalabnormalities (−5, −7) RORI Primary-Patient sample of 1.20 0.10relapsed/refractory acute myelogenous leukemia with trisomy ofchromosome #8 SOPA Primary-Patient sample of 0.10 0.09 secondary acutemyelogenous leukemia with chromsome 11q23 abnormality STGLPrimary-Patient sample of 1.88 0.62 refractory acute myelogenousleukemia with multiple chromosomal abnormalities(refractory toallogeneic BMT) A2780 Solid-Human ovarian 0.12 0.02 carcinoma A549Solid-Human lung 0.28 0.09 adenocarcinoma; G12S KRAS mutation BE(2)CSolid-human 0.07 0.04 neuroblastoma; p53 mutant CWR22 Solid-humanprostate 0.09 0.04 carcinoma FUUR1 Solid-human renal cell 0.48 0.34carcinoma with the reciprocal ASPL-TFE3 fusion transcript H11-18Solid-Human lung 0.42 0.06 adenocarcinoma; L858R EGFR mutation H1650Solid-Human lung 0.29 0.06 adenocarcinoma; DelE746- A750 EGFR mutation;wild-type RAS; PTEN null H1734 Solid-Human lung 0.24 0.04adenocarcinoma; G13C KRAS mutation H1975 Solid-Human lung 0.12 0.03adenocarcinoma; T790M/L858R EGFR mutation; R273H p53 mutation; PTENnull; wild-type KRAS H2030 Solid-human lung 0.49 0.22 adenocarcinoma;wild-type EGFR; G12C KRAS mutation; G262V P53 mutation; PTEN null H2122Solid-human lung 1.19 0.21 adenocarcinoma; G12C KRAS mutation; Q16L andC176F P53 mutations H23 Solid-Human lung 0.12 0.05 adenocarcinoma; G12cKRAS mutation; M246I P53 mutation H2444 Solid-human lung 0.31 0.03adenocarcinoma; G12V KRAS mutation H3255 Solid-Human lung 0.21 0.07adenocarcinoma; L858R EGFR mutation; wild-type KRAS; wild-type P53 H358Solid-Human lung 0.21 0.05 adenocarcinoma; G13C KRAS mutation H460Solid-human lung 0.39 0.08 adenocarcinoma; Q61H KRAS mutation H820Solid-human lung 0.09 0.05 adenocarcinoma; Del E746-E749 and T790M EGFRmutations HCC4011 Solid-Human lung 0.14 0.05 adenocarcinoma; L858R EGFRmutation; wild-type KRAS HCC827 Solid-human lung 0.28 0.12adenocarcinoma; DelE746- A750 EGFR mutation HELA N10 Solid-humancervical 1.27 0.97 adenocarcinoma (positive for human papillomavirusinfection) HPLD1 Solid-Immortalized human 0.18 0.04 bronchiolarepithelial cell with SV-40 Large T antigen HTB15 Solid-Human 0.65 0.46Glioblastoma (U-118MG), classified as Grade IV JNDSRCT1 Solid-Humandesmoplastic 0.07 0.05 small round cell tumor cell line MCF-10ASolid-human normal 0.26 0.24 mammary epithelium MCF-7 Solid-humaninvasive 0.17 0.13 breast ductal carcinoma, estrogen and progesteronereceptor positive MDA-MB-231 Solid-human metastatic 0.47 0.29 breastadenocarcinoma; estrogen, progesterone, and HER2/NEU receptor negativeMESO47 Solid-human 0.61 0.38 mesothelioma; WT-1 overexpression OVCAR3Solid-human ovarian 0.21 0.11 adenocarcinoma PC9 Solid-Human lung 0.300.07 adenocarcinoma; DelE746- A750 EGFR mutation RB355 Solid-Human 0.090.04 retinoblastoma RH30 Solid-Human 0.13 0.08 rhabdomyosarcoma; P53mutation SKMEL28 Solid-Human melanoma 0.54 0.34 SKOV3 Solid-Human 0.120.04 adenocarcinoma from the ovary derived from ascites; hypodiploidSW1736 Solid-Human anaplastic 0.16 0.02 thyroid carcinoma with BRAFV600E mutation TC71 Solid-Human Ewing's 0.15 0.09 sarcoma Y79Solid-Human 0.06 0.05 retinoblastoma

Example 3. Stability Studies Using Granaticin A

Standard liver microsome stability tests were performed with mouse andhuman microsomes with granaticin A and revealed that this drug iscleared quickly by the liver. The stability of granaticin A wasdetermined in mouse and human liver microsomes. In mouse livermicrosomes, recovery at 30 minutes was 3.68%. In mouse liver microsomes,recovery at 60 minutes was 3.84%. In human liver microsomes, recovery at30 minutes was 23.89%. In human liver microsomes, recovery at 60 minuteswas 12.01%.

Example 4. Preliminary Screening of Granaticin B

Standard solubility studies were conducted with granaticin B. In aqueoussolution, 4.0 mg/ml of granaticin B is soluble. In alcohol, >188 mg/mlof granaticin B is soluble. In methanol, >252 mg/ml of granaticin B issoluble.

Different formulations of granaticin B were also tested and PK datameasured (see Table 3).

TABLE 3 Preliminary Mouse PK Screening Granaticin B, Mouse AUC_(inf)Half-life C_(max) CL PK screening 5 mg/kg (hr-μg/mL) (t_(2/1) ^(β))(μg/mL) (mL/hr/kg) A: 1.25% of Ethanol 3532.5 6.9 2834.7 1415.4 &Tween-20 B: 0.6% GDO-12 477981.5 3445.5 2537.2 10.5 C: 5% GDP-12 2689.96.6 2449.9 1858.8 D: DMSO 2724.5 2.1 2210.9 1835.2

Other Embodiments

All patents, patent applications, and literature references cited hereinare incorporated herein by reference.

The foregoing has been a description of certain non-limiting embodimentsof the invention. Those of ordinary skill in the art will appreciatethat various changes and modifications to this description may be madewithout departing from the spirit or scope of the present invention, asdefined in the following claims.

What is claimed is:
 1. A method for treating cancer in a human subjectin need thereof, comprising administering to the human subject atherapeutically effective amount of a compound of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein the cancer is asolid tumor.
 2. The method of claim 1, wherein the cancer comprises agenetic mutation.
 3. The method of claim 2, wherein the genetic mutationcomprises a RAS mutation, an EGFR mutation, a KRAS mutation, a p53mutation, a BRAF mutation, a EVI1 mutation, a Flt-3 mutation, WT-1mutation, a cyclin D mutation, a PTEN mutation, an ABL kinase mutation,or a chromosomal abnormality.
 4. The method of claim 1, wherein thecancer is a multi-drug resistant (MDR) cancer.
 5. The method of claim 1,wherein the cancer is relapsed and/or refractory cancer.
 6. The methodof claim 1, further comprising administering at least one other therapyor therapeutic agent.
 7. The method of claim 6, wherein the at least oneother therapy is radiation.
 8. The method of claim 1, wherein thecompound is administered by systemic intravenous injection in a bolus orcontinuous infusion regimen.
 9. A method for treating cancer in a humansubject in need thereof comprising administering to the human subject atherapeutically effective amount of a compound of the Formula (II):

or a pharmaceutically acceptable salt thereof, wherein the cancer isprostate cancer, renal cell carcinoma, cervical cancer, glioblastoma,retinoblastoma, rhabdomyosarcoma, desmoplastic small round cell tumor,breast cancer, mesothelioma, or Ewing's sarcoma.
 10. The method of claim9, wherein the cancer comprises a genetic mutation.
 11. The method ofclaim 10, wherein the genetic mutation comprises a RAS mutation, an EGFRmutation, a KRAS mutation, a p53 mutation, a BRAF mutation, a EVI1mutation, a Flt-3 mutation, WT-1 mutation, a cyclin D mutation, a PTENmutation, an ABL kinase mutation, or a chromosomal abnormality.
 12. Themethod of claim 9, wherein the cancer is a multi-drug resistant (MDR)cancer.
 13. The method of claim 9, wherein the cancer is a relapsedand/or refractory cancer.
 14. The method of claim 9, further comprisingadministering at least one other therapy or therapeutic agent.
 15. Themethod of claim 14, wherein the at least one other therapy is radiation.16. The method according to claim 9, wherein the compound isadministered by systemic intravenous injection in a bolus or continuousinfusion regimen.